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CN1360630A - Glucoamylase variants - Google Patents

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CN1360630A
CN1360630A CN00810132A CN00810132A CN1360630A CN 1360630 A CN1360630 A CN 1360630A CN 00810132 A CN00810132 A CN 00810132A CN 00810132 A CN00810132 A CN 00810132A CN 1360630 A CN1360630 A CN 1360630A
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比贾恩·R·尼尔森
艾伦·斯文德森
亨里克·彼得森
杰斯珀·文德
汉尼·V·亨德里克森
托本·P·弗兰德森
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Abstract

The present invention relates to variants of a parent fungal glucoamylase, which exhibit altered properties, in particular improved thermostability and/or specific activity.

Description

葡糖淀粉酶变体Glucoamylase variants

                         发明领域Field of Invention

本发明涉及亲本AMG的特性改变的新葡糖淀粉酶变体(突变体)，所述特性改变具体是改善了热稳定性和/或提高了比活，所述变体适用于如淀粉转化，具体适于从淀粉生产葡萄糖，和醇类生产，甜味剂生产。更特定地，本发明涉及葡糖淀粉酶变体和此变体酶的应用。The present invention relates to novel glucoamylase variants (mutants) of the parent AMG with altered properties, in particular improved thermostability and/or increased specific activity, which variants are suitable for e.g. starch conversion, It is particularly suitable for the production of glucose from starch, and the production of alcohols and sweeteners. More particularly, the invention relates to glucoamylase variants and uses of such variant enzymes.

                         发明背景Background of the Invention

葡糖淀粉酶(1，4-α-D-葡聚糖葡萄糖水解酶，EC3.2.1.3)可以催化从淀粉或相关的寡糖和多糖分子的非还原末端释放D-葡萄糖。葡糖淀粉酶可从几种丝状真菌或酵母产生，其中曲霉属在商业上是最重要的。Glucoamylase (1,4-α-D-glucan glucohydrolase, EC 3.2.1.3) can catalyze the release of D-glucose from the non-reducing ends of starch or related oligo- and polysaccharide molecules. Glucoamylases can be produced from several filamentous fungi or yeasts, of which Aspergillus is the most commercially important.

在商业上，葡糖淀粉酶用于将被α-淀粉酶部分水解的玉米淀粉转化为为葡萄糖。葡萄糖进一步被葡萄糖异构酶转化为含几乎等量的葡萄糖和果糖的混合物。此混合物或果糖含量更高的混合物是通常使用于全世界商业用途的高果糖玉米浆。此糖浆是世界上由酶方法生产的最大产量的产品。在由淀粉转化为果糖中有关的三种酶是生产的最重要的工业用酶。Glucoamylases are used commercially to convert cornstarch, which is partially hydrolyzed by alpha-amylases, to glucose. Glucose is further converted by glucose isomerase into a mixture containing almost equal amounts of glucose and fructose. This blend, or a higher fructose blend, is high fructose corn syrup commonly used commercially throughout the world. This syrup is the product produced in the world in the largest volume by enzymatic methods. The three enzymes involved in the conversion of starch to fructose are the most important industrial enzymes produced.

考虑到在高果糖玉米浆生产中葡糖淀粉酶的商业用途，其中一个主要问题是葡糖淀粉酶的热稳定性相对较低。葡糖淀粉酶不象α-淀粉酶或葡萄糖异构酶一样热稳定，它最活跃和稳定的pH值比α-淀粉酶或葡萄糖异构酶要低。相应地，它必须用于较低温度和pH值的单独的容器中。Considering the commercial use of glucoamylases in high fructose corn syrup production, one of the major problems is the relatively low thermostability of glucoamylases. Glucoamylase is not as thermostable as α-amylase or glucose isomerase, and its most active and stable pH value is lower than that of α-amylase or glucose isomerase. Accordingly, it must be used in a separate container for lower temperatures and pH values.

黑曲霉的葡糖淀粉酶具有催化活性结构域(氨基酸1-440)和淀粉结合结构域(氨基酸509-616)，此二者被一段较长且高度O-糖基化的接头所分隔(Svensson等(1983)，Carlsberg Res.Commun.48，529-544，1983和(1986)，欧洲生物化学杂志(Eur.J.Biochem)，154，497-502)。泡盛曲霉X100的葡糖淀粉酶的催化活性结构域(氨基酸1-471)具有(α/α)6-折叠，其中六个保守的α→α环片段与内桶和外桶相连(Aleshin等(1992)，生物学化学杂志(J.Biol.Chem.)167，19291-19298)。葡糖淀粉酶与1-脱氧野尻霉素(Harris等(1993)，生物化学，32，1618-1626)和假四糖抑制因子阿卡波糖及D-葡萄糖-二氢阿卡波糖(Aleshin等(1996)，生物化学35，8319-8328)的复合物的晶体结构进一步与分别作为全酸和全碱的谷氨酸179和400相符合。已利用蛋白工程对这些残基在催化中的关键作用进行了研究(Sierks等(1990)，蛋白质工程(Protein Engng.)3，193-198；Frandsen等(1994)，生物化学，33，13808-13816)。在葡糖淀粉酶复合物结构中强调了在四个糖基残基结合亚点，-1，+1，+2，和+3处葡糖淀粉酶-糖的相互作用，利用定点诱变突变体结合动力学分析广泛地调查对结合和催化作用重要的残基(Sierks等(1989)，蛋白质工程，2，621-625；Sierks等(1990)，蛋白质工程，3，193-198；Berland等(1995)，生物化学，34，10153-10161；Frandsen等(1995)，生物化学，34，10162-10169。The glucoamylase from Aspergillus niger has a catalytically active domain (amino acids 1-440) and a starch-binding domain (amino acids 509-616), separated by a long, highly O-glycosylated linker (Svensson et al. (1983), Carlsberg Res. Commun. 48, 529-544, 1983 and (1986), Eur. J. Biochem, 154, 497-502). The catalytically active domain (amino acids 1-471) of the glucoamylase of Aspergillus awamori X100 has a (α/α) 6 -fold in which six conserved α→α loop segments are connected to the inner and outer barrels (Aleshin et al. (1992 ), Journal of Biological Chemistry (J.Biol.Chem.) 167, 19291-19298). Glucoamylase and 1-deoxynojirimycin (Harris et al. (1996), Biochemistry 35, 8319-8328) The crystal structure of the complex was further consistent with glutamic acid 179 and 400 as a full acid and a full base, respectively. The critical role of these residues in catalysis has been studied using protein engineering (Sierks et al. (1990), Protein Engineering (Protein Engng.) 3, 193-198; Frandsen et al. (1994), Biochemistry, 33, 13808- 13816). Glucoamylase-sugar interactions at four glycosyl residue binding subsites, -1, +1, +2, and +3, are highlighted in the structure of the glucoamylase complex, mutated using site-directed mutagenesis Binding kinetics analysis extensively surveys residues important for binding and catalysis (Sierks et al. (1989), Protein Eng, 2, 621-625; Sierks et al (1990), Protein Eng, 3, 193-198; Berland et al (1995), Biochemistry, 34, 10153-10161; Frandsen et al. (1995), Biochemistry, 34, 10162-10169.

黑曲霉葡糖淀粉酶中可以增强热稳定性的几种取代描述如下：i)取代α-螺旋甘氨酸：G137A和G139A(Chen等(1996)，蛋白质工程，9，499-505)；ii)去除脆弱的Asp-X肽键，D257E和D293E/Q(Chen等(1995)，蛋白质工程，8，575-582)；阻止在N182的脱氨基作用(Chen等(1994)，生物化学杂志，301，275-281)；iv)通过工程化方法添加二硫键，A246C(Fierobe等(1996)，生物化学，35，8698-8704；和v)在A435和S436中引入Pro残基(Li等(1997)，蛋白质工程，10，1199-1204。此外，Clark Ford于1997年10月17日发表的论文，酶工程14，北京/中国10月12-17日，1997，文摘号码：文摘第0-61页。此文摘提出在泡盛曲霉葡糖淀粉酶位置G137A，N20C/A27C，和S30P(未公开))进行突变以改进其热稳定性。Several substitutions that can enhance thermostability in Aspergillus niger glucoamylase are described as follows: i) substitution of α-helical glycine: G137A and G139A (Chen et al. (1996), Protein Engineering, 9, 499-505); ii) removal of Fragile Asp-X peptide bonds, D257E and D293E/Q (Chen et al. (1995), Protein Engineering, 8, 575-582); prevent deamination at N182 (Chen et al. (1994), J. Biological Chemistry, 301, 275-281); iv) adding disulfide bonds by engineering methods, A246C (Fierobe et al. (1996), Biochemistry, 35, 8698-8704; and v) introducing Pro residues in A435 and S436 (Li et al. ), Protein Engineering, 10, 1199-1204. In addition, Clark Ford's paper published on October 17, 1997, Enzyme Engineering 14, Beijing/China October 12-17, 1997, abstract number: abstract No. 0-61 pp. This abstract proposes mutations at positions G137A, N20C/A27C, and S30P (unpublished)) of the Aspergillus awamori glucoamylase to improve its thermostability.

有关葡糖淀粉酶的其它信息可在国际互联网主页(http：//www.public.iastate.edu/～pedro/glase/glase.html)上由Pedro M.Coutinho负责的“葡糖淀粉酶WWW网页”(最后一次更新97/10/08)披露了有关葡糖淀粉酶的信息，其中包括得自曲霉属菌株的葡糖淀粉酶。列出了在黑曲霉葡糖淀粉酶中的化学修饰和定点修饰。Additional information on glucoamylases is available on the Internet home page (http://www.public.iastate.edu/~pedro/glase/glase.html) at the "Glucoamylases WWW Web Page" by Pedro M. Coutinho " (last updated 97/10/08) discloses information on glucoamylases, including glucoamylases from Aspergillus strains. Chemical and site-directed modifications in Aspergillus niger glucoamylase are listed.

                        发明简述Brief description of the invention

本发明的目的是提供适用于，例如淀粉转化过程中的糖化步骤的葡糖淀粉酶变体。It is an object of the present invention to provide glucoamylase variants suitable for use eg in the saccharification step of starch conversion processes.

术语“热稳定的葡糖淀粉酶变体”在本发明中指与相应的亲本葡糖淀粉酶相比有较高T1/2(半衰期)的葡糖淀粉酶变体。在下面的“材料和方法”部分描述了T1/2的测定(方法I和方法II)。The term "thermostable glucoamylase variant" refers in the present invention to a glucoamylase variant which has a higher T 1/2 (half-life) compared to the corresponding parent glucoamylase. Determination of T 1/2 (Method I and Method II) is described in the "Materials and Methods" section below.

术语“比活提高的葡糖淀粉酶变体”在本发明中指提高了对所述糖分子中α-1，4键的比活。比活测定为kcat或AGU/mg(按下文“材料和方法”部分所述测定)。提高比活意味着kcat或AGU/mg值分别相对于相应地亲本葡糖淀粉酶有了提高。The term "glucoamylase variant with increased specific activity" refers in the present invention to increased specific activity towards the α-1,4 bond in said sugar molecule. Specific activity was determined as k cat or AGU/mg (determined as described in the "Materials and Methods" section below). Increased specific activity means that the value of k cat or AGU/mg, respectively, is increased relative to the corresponding parent glucoamylase.

本发明人提供了亲本葡糖淀粉酶的几种改进了热稳定性和/或提高了比活的变体。热稳定性的改进可通过突变，例如在亲本葡糖淀粉酶所选位置上的取代和/或缺失，插入来实现。下面有对此的详述。The inventors have provided several variants of the parent glucoamylase with improved thermostability and/or increased specific activity. Improvements in thermostability can be achieved by mutations, such as substitutions and/or deletions, insertions at selected positions in the parent glucoamylase. This is detailed below.

命名法nomenclature

在本说明书和权利要求书中，使用传统的一字母和三字母代码表示氨基酸残基。In the specification and claims, amino acid residues are referred to using the conventional one-letter and three-letter codes.

为便于引用，本发明的AMG变体采用下述的命名法：For ease of reference, the AMG variants of the present invention employ the following nomenclature:

原氨基酸：位置：用于取代的氨基酸Original Amino Acid: Position: Amino Acid for Substitution

根据该命名法，用天冬酰胺取代第30位的丙氨酸示为：Ala30Asn或A30N，在相同位置处缺失丙氨酸示为：Ala30*或A30*，而插入另一个氨基酸残基，如赖氨酸示为：Ala30AlaLys或A30AK。缺失一段连续的氨基酸残基，如氨基酸残基30-33示为(30-33)*或Δ(A30-N33)。According to this nomenclature, substitution of an alanine at position 30 with an asparagine is shown as: Ala30Asn or A30N, deletion of an alanine at the same position is shown as: Ala30 * or A30 * , and insertion of another amino acid residue, as in Lysine is shown as: Ala30AlaLys or A30AK. A stretch of consecutive amino acid residues is deleted, such as amino acid residues 30-33 are shown as (30-33) * or Δ(A30-N33).

当特定的AMG相对于其它AMG而言含有“缺失”，并在该位置插入某个氨基酸，例如在第36位插入天冬氨酸时，示为*36Asp或*36D。When a particular AMG contains a "deletion" relative to other AMGs and an amino acid is inserted at that position, for example aspartic acid at position 36, it is indicated as * 36Asp or * 36D.

多个突变由加号隔开，即：Ala30Asp+Glu34Ser或A30N+E34S表示分别将第30和34位的丙氨酸和谷氨酸用天冬酰胺和丝氨酸取代。多个突变也可以如下隔开，其意义与加号相同：Ala30Asp/Glu34Ser或A30N/E34S。Multiple mutations are separated by plus signs, namely: Ala30Asp+Glu34Ser or A30N+E34S means that alanine and glutamic acid at positions 30 and 34 are replaced with asparagine and serine, respectively. Multiple mutations can also be separated as follows, with the same meaning as a plus sign: Ala30Asp/Glu34Ser or A30N/E34S.

当在给定的位置插入一个或多个其它氨基酸残基时，示为A30N，E或A30N/E或A30N或A30E。When one or more other amino acid residues are inserted at a given position, it is indicated as A30N, E or A30N/E or A30N or A30E.

另外，当本文鉴定出适于修饰的位置，而没有暗示任何具体的修饰时，应理解为可用任一氨基酸残基取代该位置存在的氨基酸残基。因此，例如，当提到修饰第30位的丙氨酸，但未具体说明时，应理解为丙氨酸可缺失，或用任一其它氨基酸，即下列任一氨基酸所取代：R，N，D，A，C，Q，E，G，H，I，L，K，M，F，P，S，T， W，Y，V。Additionally, when a position suitable for modification is identified herein without implying any specific modification, it is understood that any amino acid residue may be substituted for the amino acid residue present at that position. Thus, for example, when reference is made to modifying the alanine at position 30, but not specifically stated, it is understood that the alanine may be deleted, or substituted with any other amino acid, namely any of the following: R, N, D, A, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, V.

                      附图简述Brief description of attached drawings

图1表示了含有黑曲霉G1葡糖淀粉酶基因的质粒pCAMG91。Figure 1 shows plasmid pCAMG91 containing the A. niger G1 glucoamylase gene.

                      发明详述                    Invention Details

本发明旨在改进特定葡糖淀粉酶的热稳定性和/或提高其比活，所述酶得自真菌生物，特别是黑曲霉属的菌株这些酶本身已根据它们在诸如淀粉转化或乙醇发酵中的适当性质进行了筛选。The present invention aims at improving the thermostability and/or increasing the specific activity of specific glucoamylases obtained from fungal organisms, in particular strains of the genus Aspergillus niger which have themselves been based on their use in processes such as starch conversion or ethanol fermentation Appropriate properties were screened.

本发明人从中惊奇地发现通过修饰亲本葡糖淀粉酶的氨基酸序列上的一或多个氨基酸残基确实有可能改进亲本葡糖淀粉酶热稳定性和/或提高比活。本发明就是基于此发现。The inventors have surprisingly found that it is indeed possible to improve the thermal stability and/or increase the specific activity of the parent glucoamylase by modifying one or more amino acid residues in the amino acid sequence of the parent glucoamylase. The present invention is based on this discovery.

因此，本发明的第一个方面涉及亲本葡糖淀粉酶在下述位置包含一或多个突变的变体。Accordingly, a first aspect of the invention relates to variants of the parent glucoamylase comprising one or more mutations at the positions described below.

亲本葡糖淀粉酶parent glucoamylase

本发明考虑的亲本葡糖淀粉酶包括野生型葡糖淀粉酶，真菌葡糖淀粉酶，特别是得自曲霉属菌株，如黑曲霉或泡盛曲霉的真菌葡糖淀粉酶及其变体或突变体，同源葡糖淀粉酶，和其它与SEQ ID NO：2结构和/或功能相似的葡糖淀粉酶。特别予以考虑的是公开于Boel等(1984)，“从两个不同但密切相关的mRNAs合成的黑曲霉葡糖淀粉酶G1和G2”EMBO J.3(5)，p.1097-1102中的黑曲霉葡糖淀粉酶G1和G2。G2葡糖淀粉酶公开为SEQ IDNO：2。在另一实施方案中，AMG骨架得自踝节菌属，特别是公开于WO99/28448的T.emersonii(参见WO 99/28448的SEQ ID NO：7)。Parent glucoamylases contemplated by the present invention include wild-type glucoamylases, fungal glucoamylases, especially fungal glucoamylases obtained from strains of the genus Aspergillus, such as Aspergillus niger or Aspergillus awamori, and variants or mutants thereof , homologous glucoamylases, and other glucoamylases similar in structure and/or function to SEQ ID NO:2. Particularly contemplated are those disclosed in Boel et al. (1984), "Aspergillus niger glucoamylase G1 and G2 synthesized from two distinct but closely related mRNAs" EMBO J.3(5), p.1097-1102. Aspergillus niger glucoamylases G1 and G2. The G2 glucoamylase is disclosed as SEQ ID NO:2. In another embodiment, the AMG backbone is obtained from Talaromyces, in particular T. emersonii disclosed in WO 99/28448 (see SEQ ID NO: 7 of WO 99/28448).

市售亲本葡糖淀粉酶Commercially available parent glucoamylase

考虑的市售亲本葡糖淀粉酶包括Novo Nordisk的AMG，以及Genencor，Inc.USA和Gist-Brocades，Delft，The Netherlands的葡糖淀粉酶。Commercially available parent glucoamylases considered include AMG from Novo Nordisk, and glucoamylases from Genencor, Inc. USA and Gist-Brocades, Delft, The Netherlands.

本发明的葡糖淀粉酶变体Glucoamylase variants of the invention

在第一个方面，本发明涉及亲本葡糖淀粉酶的变体，其包括在下列一或多个位置的改变：59，66，72，119，189，223，227，313，340，342，352，379，386，393，395，402，408，416，425，427，444，486，490，494，In a first aspect, the invention relates to variants of the parent glucoamylase comprising changes at one or more of the following positions: 59, 66, 72, 119, 189, 223, 227, 313, 340, 342, 352, 379, 386, 393, 395, 402, 408, 416, 425, 427, 444, 486, 490, 494,

其中(a)所述每一改变是where each change described in (a) is

(i)在占据此位置的氨基酸下游插入氨基酸，(i) an amino acid is inserted downstream of the amino acid occupying that position,

(ii)占据此位置的氨基酸的缺失，或(ii) a deletion of the amino acid occupying this position, or

(iii)占据此位置的氨基酸用另一氨基酸取代，(iii) the amino acid occupying this position is substituted with another amino acid,

(b)所述变体具有亲本葡糖淀粉酶活性且(c)每一位置对应于具有SEQID NO：2之氨基酸序列的亲本葡糖淀粉酶氨基酸序列的位置。(b) the variant has parental glucoamylase activity and (c) each position corresponds to the position of the parental glucoamylase amino acid sequence having the amino acid sequence of SEQ ID NO:2.

此外，本发明涉及这样一种葡糖淀粉酶变体，其亲本葡糖淀粉酶具有与SEQ ID NO：2之氨基酸序列至少约65％，优选至少约70％，更优选至少约80％，更优选至少约90％，最优选至少约95％，还最优选至少约97％同一性的氨基酸序列。In addition, the present invention relates to such a glucoamylase variant, its parent glucoamylase has at least about 65%, preferably at least about 70%, more preferably at least about 80%, more preferably at least about 80% of the amino acid sequence of SEQ ID NO: 2 Amino acid sequences that are at least about 90%, most preferably at least about 95%, and most preferably still at least about 97% identical are preferred.

本发明还涉及亲本葡糖淀粉酶的下述变体，其包含一或多个下列改变：V59A，L66V/R，T72I，Sll9P，I189T，Y223F，F227Y，N313G，S340G，E342A，R，D，N，C，Q，G，H，I，L，K，M，F，P，S，T，W，Y，V，优选E342T，K352R，S356G，T379A，S386K，N，R，P，A393R，S395R，Y402F，E408R，T416A，R，D，N，C，Q，G，H，I，L，K，M，F，P，S，E，W，Y，V，优选T416H，A425T，N427S/M，S444G，S486G，T490A，T494P/A，其中(a)所述变体具有葡糖淀粉酶活性且(b)每一位置对应于具有SEQ ID NO：2之氨基酸序列的亲本葡糖淀粉酶的氨基酸序列中位置。The present invention also relates to the following variants of the parent glucoamylase comprising one or more of the following changes: V59A, L66V/R, T72I, S119P, I189T, Y223F, F227Y, N313G, S340G, E342A, R, D, N, C, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V, preferably E342T, K352R, S356G, T379A, S386K, N, R, P, A393R , S395R, Y402F, E408R, T416A, R, D, N, C, Q, G, H, I, L, K, M, F, P, S, E, W, Y, V, preferably T416H, A425T, N427S/M, S444G, S486G, T490A, T494P/A, wherein (a) the variant has glucoamylase activity and (b) each position corresponds to the parent glucose having the amino acid sequence of SEQ ID NO:2 position in the amino acid sequence of the amylase.

特定的突变组合包括：Specific combinations of mutations include:

L66R+Y402F+N427S+S486G+A1V；N427M+S44G+V470M+T2K+S30P；L66R+Y402F+N427S+S486G+A1V; N427M+S44G+V470M+T2K+S30P;

T416H+Y402F+312Q+S119P；A425T+E408R+E386K+A495T；T416H+Y402F+312Q+S119P; A425T+E408R+E386K+A495T;

T379A+T2E+S386K+A393R；S386N+E408R；L66V+T2R+S394P+Y402F+RL；T379A+T2E+S386K+A393R; S386N+E408R; L66V+T2R+S394P+Y402F+RL;

S386R+T2R+A393R；I189T+Y223F+F227Y+S119P+Y402F；S386R+T2R+A393R; I189T+Y223F+F227Y+S119P+Y402F;

S386P+S340G+D357S+T360V；  V59A+S119P；  V59A+N313G；  V59A+A393R；S386P+S340G+D357S+T360V; V59A+S119P; V59A+N313G; V59A+A393R;

V59A+Y402F； V59A+E408R；   V59A+S119P+N313G；  V59A+N313G+A393R；V59A+Y402F; V59A+E408R; V59A+S119P+N313G; V59A+N313G+A393R;

V59A+A393R+Y402F； V59A+Y402F+E408R；    V59A+S119P+N313G+A393R；V59A+A393R+Y402F; V59A+Y402F+E408R; V59A+S119P+N313G+A393R;

V59A+N313G+A393R+Y402F；                 V59A+A393R+Y402F+E408R；V59A+N313G+A393R+Y402F; V59A+A393R+Y402F+E408R;

V59A+S119P+N313G+A393R+Y402F；   V59A+N313G+A393R+Y402F+E408R；V59A+S119P+N313G+A393R+Y402F; V59A+N313G+A393R+Y402F+E408R;

V59A+S119P+L66R；     V59A+S119P+S340G；     V59A+S119P+S395R；V59A+S119P+L66R; V59A+S119P+S340G; V59A+S119P+S395R;

V59A+S119P+L66R+S340G；                V59A+S119P+S340G+S395R；V59A+S119P+L66R+S340G; V59A+S119P+S340G+S395R;

V59A+S119P+S395R+L66R；           V59A+S119P+S395R+L66R+S340G；V59A+S119P+S395R+L66R; V59A+S119P+S395R+L66R+S340G;

V59A+N313G+L66R；      V59A+N313G+S340G；    V59A+N313G+S395R；V59A+N313G+L66R; V59A+N313G+S340G; V59A+N313G+S395R;

V59A+N313G+L66R+S340G；V59A+N313G+S340G+S395R；V59A+N313G+L66R+S340G; V59A+N313G+S340G+S395R;

V59A+N313G+S395R+L66R；            V59A+N313G+S395R+L66R+S340G；V59A+N313G+S395R+L66R; V59A+N313G+S395R+L66R+S340G;

V59A+A393R+L66R；      V59A+A393R+S340G；     V59A+A393R+S395R；V59A+A393R+L66R+S340G；                     V59A+A393R+S340G+S395R；V59A+A393R+S395R+L66R+S340G；                      V59A+Y402F+L66R；V59A+Y402F+S340G；  V59A+Y402F+S395R；      V59A+Y402F+L66R+S395R；V59A+Y402F+L66R+S340G；V59A+Y402F+L66R+S395R+S340G；V59A+E408R+L66R；V59A+E408R+S395R；V59A+E408R+S340G；V59A+E408R+S395R+S340G；V59A+E408R+L66R+S340G；V59A+E408R+L66R+S395R；V59A+E408R+L66R+S395R+S340G；V59A+S119P+N313G+L66R；V59A+S119P+N313G+L66R+S340G；V59A+S119P+N313G+L66R+S395R；V59A+S119P+N313G+L66R+S395R+S340G；V59A+N313G+A393R+L66R；V59A+N313G+A393R+L66R+S395R； V59A+N313G+A393R+L66R+S340G；V59A+N313G+A393R+ L66R+S340G+S395R； V59A+A393R+Y402F；V59A+Y402F+E408R；V59A+S119P+N313G+A393R；V59A+N313G+A393R+Y402F；V59A+A393R+Y402F+E408R；V59A+S119P+N313G+A393R+Y402F；V59A+N313G+A393R+Y402F+E408R；S119P+N313G；N313G+A393R；A393R+Y402F；Y402F+E408R；S119P+N313G+A393R；N313G+A393R+Y402F；A393R+Y402F+E408R；V59A+S119P+N313G+A393R+Y402F；N313G+A393R+Y402F+E408R；S119P+L66R；V59A+S119P+S340G；S119P+S395R；S119P+L66R+S340G；S119P+S340G+S395R；S119P+S395R+L66R；S119P+S395R+L66R+S340G；N313G+L66R；N313G+S340G；N313G+S395R；N313G+L66R+S340G；N313G+S340G+S395R；N313G+S395R+L66R；N313G+S395R+L66R+S340G；A393R+L66R；A393R+S340G；A393R+S395R；A393R+L66R+S340G；A393R+S340G+S395R；A393R+S395R+L66R+S340G；Y402F+L66R；Y402F+S340G；Y402F+S395R；Y402F+L66R+S395R；Y402F+L66R+S340G；Y402F+L66R+S395R+S340G；E408R+L66R；E408R+S395R；E408R+S340G；E408R+S395R+S340G；E408R+L66R+S340G；E408R+L66R+S395R；E408R+L66R+S395R+S340G；S119P+N313G+L66R；S119P+N313G+L66R+S340G；S119P+N313G+L66R+S395R；S119P+N313G+L66R+S395R+S340G；N313G+A393R+L66R；N313G+A393R+L66R+S395R；N313G+A393R+ L66R+S340G；N313G+A393R+L66R+S340G+S395R；A393R+Y402F；Y402F+E408R；V59A+S119P+N313G+A393R；N313G+A393R+Y402F；A393R+Y402F+E408R；S119P+N313G+A393R+Y402F；N313G+A393R+Y402F+E408R.V59A+S119P+S340G；S119P+S395R；S119P+S340G；S119P+S340G+S395R；S119P+S395R；S119P+S395R+S340G；N313G+S340G；N313P+S395R；313G+S340G； N313G+S395R； N313G+S395R+S340G； A393R+S340G；A393R+S395R+S340G；Y402F+S395R；Y402F+S340G；Y402F+S395R+S340G；E408R+S340G；E408R+S395R；E408R+S395R+S340G；S119P+N313G；S119P+N313G+S340G；S119P+N313G+S395R；S119P+N313G+S395R+S340G；N313G+A393R；N313G+A393R+S395R；N313G+A393R+S340G；N313G+A393R+S340G+S395R；  .N313G+A393R；V59A+N313G+A393R+Y402F；V59A+S340G； L66R+S340G；S340G+S395R；S395R+L66R；S395R+L66R+S340G；N313G+L66R；N313G+L66R+S340G；N313G+L66R+S395R；N313G+L66R+S395R+S340G；V59A+N313G+A393R；N313G+A393R+Y402F；S119P+A393R；A393R+Y402F；V59A+S119P+A393R+Y402F；A393R+Y402F+E408R；S119P+S395R+L66R+S340G；L66R+S340G；S340G+S395R；S395R+L66R；S395R+L66R+S340G；S119P+L66R；S119P+L66R+S340G；S119P+L66R+S395R；S119P+L66R+S395R+S340G；A393R+ L66R；A393R+L66R+S395R；A393R+ L66R+S340G；A393R+L66R+S340G+S395R；V59A+S119P+A393R；A393R+Y402F；S119P+A393R+Y402F；A393R+Y402F+E408R；S119P+N313G；N313G+Y402F；Y402F+E408R；V59A+S119P+N313G+Y402F；N313G+Y402F+E408R；L66R+S340G；S340G+S395R；S395R+L66R+S340G；N313G+L66R；N313G+L66R+S395R；N313G+L66R+S340G；N313G+L66R+S340G+S395R；V59A+S119P+N313G；N313G+Y402F；Y402F+E408R；S119P+N313G+Y402F；N313G+Y402F+E408R.S119P+S340G；S119P+L66R；S119P+L66R+S340G；N313G+S340G；N313G+L66R；N313G+L66R+S340G；A393R+S340G；A393R+L66R+S340G；Y402F+L66R；Y402F+L66R+S340G；Y402F+L66R+S340G；E408R+S340G；E408R+L66R；E408R+L66R+S340G；S119P+N313G+L66R；S119P+N313G+L66R+S340G；N313G+A393R+L66R；N313G+A393R+ L66R+S340G；N313G+A393R；A393R+E408R；V59A+S119P+N313G+A393R；N313G+A393R+E408R；L66R+S395R；L66R+S340G；L66R+S395R+S340G；N313G+A393R；A393R+E408R；S119P+N313G+A393R；N313G+A393R+E408R.A393R+Y402F；N313G+A393R+Y402F；S395R+S340G； L66R+S340G；L66R+S395R；L66R+S395R+S340G； A393R+Y402F；N313G+A393R+Y402F.S119P+L66R；V59A+S119P；S119P+S395R；S119P+L66R；S119P+S395R；V59A+A393R+L66R；      V59A+A393R+S340G；     V59A+A393R+S395R；V59A+A393R+L66R+S340G；                     V59A+A393R+S340G+S395R；V59A+A393R+S395R+L66R+S340G；                      V59A+Y402F+L66R； V59A+Y402F+S340G; V59A+Y402F+S395R; V59A+Y402F+L66R+S395R; V59A+Y402F+L66R+S340G; V59A+E408R+S340G; V59A+E408R+S395R+S340G; V59A+E408R+L66R+S340G; V59A+E408R+L66R+S395R; V59A+E408R+L66R+S395R+S340G; S119P+N313G+L66R+S340G; V59A+S119P+N313G+L66R+S395R; V59A+S119P+N313G+L66R+S395R+S340G; V59A+N313G+A393R+L66R; N313G+A393R+L66R+S340G；V59A+N313G+A393R+ L66R+S340G+S395R； V59A+A393R+Y402F；V59A+Y402F+E408R；V59A+S119P+N313G+A393R；V59A+N313G+A393R+Y402F；V59A+A393R +Y402F+E408R; V59A+S119P+N313G+A393R+Y402F; V59A+N313G+A393R+Y402F+E408R; S119P+N313G; N313G+A393R; A393R+Y402F; +Y402F; A393R+Y402F+E408R; V59A+S119P+N313G+A393R+Y402F; N313G+A393R+Y402F+E408R; S119P+L66R; V59A+S119P+S340G; +S395R; S119P+S395R+L66R; S119P+S395R+L66R+S340G; N313G+L66R; +L66R+S340G; A393R+L66R; A393R+S340G; A393R+S395R; A393R+L66R+S340G; A393R+S340G+S395R; +L66R+S395R; Y402F+L66R+S340G; Y402F+L66R+S395R+S340G; E408R+L66R; +L66R+S395R+S340G; S119P+N313G+L66R; S119P+N313G+L66R+S340G; S119P+N313G+L66R+S395R; S119P+N313G+L66R+S395R+S340G; +S395R；N313G+A393R+ L66R+S340G；N313G+A393R+L66R+S340G+S395R；A393R+Y402F；Y402F+E408R；V59A+S119P+N313G+A393R；N313G+A393R+Y402F；A393R+Y402F+E408R；S119P+ N313G+A393R+Y402F；N313G+A393R+Y402F+E408R.V59A+S119P+S340G；S119P+S395R；S119P+S340G；S119P+S340G+S395R；S119P+S395R；S119P+S395R+S340G；N313G+S340G；N313P+ S395R；313G+S340G； N313G+S395R； N313G+S395R+S340G； A393R+S340G；A393R+S395R+S340G；Y402F+S395R；Y402F+S340G；Y402F+S395R+S340G；E408R+S340G；E408R+S395R；E408R+ S395R+S340G；S119P+N313G；S119P+N313G+S340G；S119P+N313G+S395R；S119P+N313G+S395R+S340G；N313G+A393R；N313G+A393R+S395R；N313G+A393R+S340G；N313G+A393R+S340G+ S395R; .N313G+A393R; V59A+N313G+A393R+Y402F; V59A+S340G; L66R+S340G; S340G+S395R; S395R+L66R; +S395R; N313G+L66R+S395R+S340G; V59A+N313G+A393R; N313G+A393R+Y402F; S119P+A393R; +S340G; L66R+S340G; S340G+S395R; S395R+L66R; S395R+L66R+S340G; S119P+L66R; L66R+S395R；A393R+ L66R+S340G；A393R+L66R+S340G+S395R；V59A+S119P+A393R；A393R+Y402F；S119P+A393R+Y402F；A393R+Y402F+E408R；S119P+N313G；N313G+Y402F；Y402F+E408R ;V59A+S119P+N313G+Y402F; N313G+Y402F+E408R; L66R+S340G; +S395R; V59A+S119P+N313G; N313G+Y402F; Y402F+E408R; S119P+N313G+Y402F; ;N313G+L66R+S340G; A393R+S340G; A393R+L66R+S340G; Y402F+L66R; Y402F+L66R+S340G; Y402F+L66R+S340G; +L66R; S119P+N313G+L66R+S340G; N313G+A393R+L66R; N313G+A393R+ L66R+S340G; L66R+S340G; L66R+S395R+S340G; N313G+A393R; A393R+E408R; S119P+N313G+A393R; S395R; L66R+S395R+S340G; A393R+Y402F; N313G+A393R+Y402F.S119P+L66R; V59A+S119P; S119P+S395R; S119P+L66R; S119P+S395R;

S119P+S395R+L66R；N313G+L66R；N313G+S395R；N313G+S395R+L66R；S119P+S395R+L66R; N313G+L66R; N313G+S395R; N313G+S395R+L66R;

A393R+L66R；A393R+S395R；A393R+S395R+L66R；Y402F+L66R；A393R+L66R; A393R+S395R; A393R+S395R+L66R; Y402F+L66R;

Y402F+L66R+S395R；E408R+S395R；E408R+L66R；E408R+L66R+S395R；Y402F+L66R+S395R; E408R+S395R; E408R+L66R; E408R+L66R+S395R;

S119P+N313G+L66R；S119P+N313G+L66R+S395R；N313G+A393R+L66R；S119P+N313G+L66R; S119P+N313G+L66R+S395R; N313G+A393R+L66R;

N313G+A393R+L66R+S39SR；N313G+A393R+L66R+S39SR;

N9A+S56A+V59A+S119P+A246T+N313G+E342T+A393R+S394R+Y402F+E408R；N9A+S56A+V59A+S119P+A246T+N313G+E342T+A393R+S394R+Y402F+E408R;

S56A+V59A+S119P+A246T+N313G+E342T+A393R+S394R+Y402F+E408R；S56A+V59A+S119P+A246T+N313G+E342T+A393R+S394R+Y402F+E408R;

V59A+S119P+A246T+N313G+E342T+A393R+S394R+Y402F+E408R；V59A+S119P+A246T+N313G+E342T+A393R+S394R+Y402F+E408R;

S119P+A246T+N313G+E342T+A393R+S394R+Y402F+E408R；S119P+A246T+N313G+E342T+A393R+S394R+Y402F+E408R;

A246T+N313G+E342T+A393R+S394R+Y402F+E408R；A246T+N313G+E342T+A393R+S394R+Y402F+E408R;

N313G+E342T+A393R+S394R+Y402F+E408R；N313G+E342T+A393R+S394R+Y402F+E408R;

E342T+A393R+S394R+Y402F+E408R；E342T+A393R+S394R+Y402F+E408R;

A393R+S394R+Y402F+E408R；S394R+Y402F+E408R；Y402F+E408R；A393R+S394R+Y402F+E408R; S394R+Y402F+E408R; Y402F+E408R;

V59A+L66R+T72I+S119P+N313G+S340G+S356G+A393R+Y402F+E408R+N427MV59A+L66R+T72I+S119P+N313G+S340G+S356G+A393R+Y402F+E408R+N427M

；;

L66R+T72I+S119P+N313G+S340G+S356G+A393R+Y402F+E408R+N427M；L66R+T72I+S119P+N313G+S340G+S356G+A393R+Y402F+E408R+N427M;

T72I+S119P+N313G+S340G+S356G+A393R+Y402F+E408R+N427M；T72I+S119P+N313G+S340G+S356G+A393R+Y402F+E408R+N427M;

S119P+N313G+S340G+S356G+A393R+Y402F+E408R+N427M；S119P+N313G+S340G+S356G+A393R+Y402F+E408R+N427M;

N313G+S340G+S356G+A393R+Y402F+E408R+N427M；N313G+S340G+S356G+A393R+Y402F+E408R+N427M;

S340G+S356G+A393R+Y402F+E408R+N427M；S340G+S356G+A393R+Y402F+E408R+N427M;

S356G+A393R+Y402F+E408R+N427M；A393R+Y402F+E408R+N427M；S356G+A393R+Y402F+E408R+N427M; A393R+Y402F+E408R+N427M;

Y402F+E408R+N427M；E408R+N427M；Y402F+E408R+N427M; E408R+N427M;

I189T+Y223F+F227Y+S119P+Y402F；Y223F+F227Y+S119P+Y402F；I189T+Y223F+F227Y+S119P+Y402F; Y223F+F227Y+S119P+Y402F;

F227Y+S119P+Y402F；S119P+Y402F；I189T+Y223F+F227Y+Y402F；F227Y+S119P+Y402F; S119P+Y402F; I189T+Y223F+F227Y+Y402F;

I189T+Y223F+F227Y；I189T+Y223F；I189T+F227Y；I189T+F227Y+S119P；I189T+Y223F+F227Y; I189T+Y223F; I189T+F227Y; I189T+F227Y+S119P;

I189T+F227Y+Y402F；Y223F+F227Y+Y402F；Y223F+F227Y+S119P.I189T+F227Y+Y402F; Y223F+F227Y+Y402F; Y223F+F227Y+S119P.

本发明还涉及这样一种葡糖淀粉酶变体，其亲本酶由在中度，优选在高度严谨条件下与SEQ ID NO：1的核酸序列或其互补链杂交的核酸序列编码。The present invention also relates to such a glucoamylase variant, whose parent enzyme is encoded by a nucleic acid sequence that hybridizes to the nucleic acid sequence of SEQ ID NO: 1 or its complementary chain under moderate, preferably highly stringent conditions.

改进的热稳定性Improved Thermal Stability

在另一方面，本发明涉及亲本葡糖淀粉酶改进了热稳定性的变体，具体是在40-80℃，优选63-75℃，具体在pH4-5，使用麦芽糖糊精为底物时改进了热稳定性，所述变体在表示为SEQ ID NO：2的氨基酸序列的下列位置含有一或多个突变：59，66，72，119，189，223，227，313，340，342，352，379，386，393，395，402，408，416，425，427，444，486，490，494，或是与SEQ ID NO：2的氨基酸序列有至少60％同源性的葡糖淀粉酶中相应位置。In another aspect, the present invention relates to variants of the parent glucoamylase with improved thermostability, in particular at 40-80°C, preferably 63-75°C, in particular at pH 4-5, using maltodextrin as substrate Improved thermostability, said variant containing one or more mutations at the following positions of the amino acid sequence represented as SEQ ID NO: 2: 59, 66, 72, 119, 189, 223, 227, 313, 340, 342 , 352, 379, 386, 393, 395, 402, 408, 416, 425, 427, 444, 486, 490, 494, or glucose having at least 60% homology to the amino acid sequence of SEQ ID NO: 2 Corresponding position in amylase.

可改进热稳定性的特定取代包括：V59A，L66V/R，T72I，S119P，I189T，Y223F，F227Y，N313G，S340G，E342A，R，D，N，C，Q，G，H，I，L，K，M，F，P，S，T，W，Y，V，优选E342T，K352R，S356G，T379A，S386K，N，R，P，A393R，S395R，Y402F，E408R，T416A，R，D，N，C，Q，G，H，I，L，K，M，F，P，S，E，W，Y，V，优选T416H，A425T，N427S/M，S444G，S486G，T490A，T494P/A。Specific substitutions that may improve thermal stability include: V59A, L66V/R, T72I, S119P, I189T, Y223F, F227Y, N313G, S340G, E342A, R, D, N, C, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V, preferably E342T, K352R, S356G, T379A, S386K, N, R, P, A393R, S395R, Y402F, E408R, T416A, R, D, N , C, Q, G, H, I, L, K, M, F, P, S, E, W, Y, V, preferably T416H, A425T, N427S/M, S444G, S486G, T490A, T494P/A.

具体的突变组合包括：Specific combinations of mutations include:

E408R+A425T+S465P+T494A，E408R+A425T+S465P+T494A,

A425T+E408R+S386K+A495T，A425T+E408R+S386K+A495T,

T379A+T2E+S386K+A393R，T379A+T2E+S386K+A393R,

S386N+E408R，S386N+E408R,

L66V+T2R+S394P+Y402F+RL N-末端延伸)L66V+T2R+S394P+Y402F+RL N-terminal extension)

S386R+T2R+A393R.S386R+T2R+A393R.

N427S+S486G+A1V+L66R+Y402F，N427S+S486G+A1V+L66R+Y402F,

N427M+S44G+V470M+T2K+S30P，N427M+S44G+V470M+T2K+S30P,

T490A+V59A++A393R+PLASD(N-末端延伸)T490A+V59A++A393R+PLASD (N-terminal extension)

在“本发明葡糖淀粉酶变体”部分列出的所有变体都被认为具有改进的热稳定性。实施例2和4中显示了本发明所选变体的这方面。All variants listed in the section "Glucoamylase variants of the invention" are believed to have improved thermostability. This aspect of selected variants of the invention is shown in Examples 2 and 4.

提高的比活improved specific activity

在另一方面，本发明涉及亲本葡糖淀粉酶比活已提高的变体，所述变体在表示为SEQ ID NO：2的氨基酸序列的下列位置含有一或多个突变：59，66，72，119，189，223，227，313，340，342，352，379，386，393，395，402，408，416，425，427，444，486，490，494，优选189，223，227或与SEQ ID NO：2的氨基酸序列有至少60％同源性的葡糖淀粉酶中相应位置。In another aspect, the present invention relates to variants with increased specific activity of the parent glucoamylase, said variants containing one or more mutations at the following positions of the amino acid sequence represented as SEQ ID NO: 2: 59, 66, 72, 119, 189, 223, 227, 313, 340, 342, 352, 379, 386, 393, 395, 402, 408, 416, 425, 427, 444, 486, 490, 494, preferably 189, 223, 227 Or the corresponding position in a glucoamylase having at least 60% homology to the amino acid sequence of SEQ ID NO:2.

可提高比活的具体突变包括：V59A，L66V/R，T72I，S119P，I189T，Y223F，F227Y，N313G，S340G，K352R，S356G，T379A，S386K，N，R，P，A393R，S395R，Y402F，E408R，T416A，R，D，N，C，Q，G，H，I，L，K，M，F，P，S，E，W，Y，V优选T416H，A425T，N427S/M，S444G，S486G，T490A，T494P/A，优选I189T，Y223F，F227Y。Specific mutations that increase specific activity include: V59A, L66V/R, T72I, S119P, I189T, Y223F, F227Y, N313G, S340G, K352R, S356G, T379A, S386K, N, R, P, A393R, S395R, Y402F, E408R , T416A, R, D, N, C, Q, G, H, I, L, K, M, F, P, S, E, W, Y, V preferably T416H, A425T, N427S/M, S444G, S486G , T490A, T494P/A, preferably I189T, Y223F, F227Y.

具体的突变组合包括：Specific combinations of mutations include:

I189T+Y223F+F227Y+S119F+Y402P；I189T+Y223F+F227Y+S119F+Y402P;

Y223F+F227Y+S119P+Y402F；F227Y+S119P+Y402F；S119P+Y402F；Y223F+F227Y+S119P+Y402F; F227Y+S119P+Y402F; S119P+Y402F;

I189T+Y223F+F227Y+Y402F；I189T+Y223F+F227Y；I189T+Y223F；I189T+Y223F+F227Y+Y402F; I189T+Y223F+F227Y; I189T+Y223F;

I189T+F227Y；I189T+F227Y+S119P；I189T+F227Y+Y402F；I189T+F227Y; I189T+F227Y+S119P; I189T+F227Y+Y402F;

Y223F+F227Y+Y402F；Y223F+F227Y+S119P.Y223F+F227Y+Y402F; Y223F+F227Y+S119P.

在“本发明葡糖淀粉酶变体”部分列出的所有变体都被认为具有已提高的比活。实施例3中显示了本发明所选变体的这方面。All variants listed in the section "Glucoamylase variants of the invention" are considered to have improved specific activities. This aspect of selected variants of the invention is shown in Example 3.

同源性(同一性)Homology (identity)

针对上述亲本葡糖淀粉酶的同源性测定为两个蛋白序列之间可指示其中第一个序列衍生自第二个序列的同一性程度。此同源性适于由本领域所熟知的计算机程序测定，如GCG软件包提供的GAP(Wisconsin软件包程序手册，第8版，1994.8，遗传学计算机组，575 Science Drive，Madison，Wisconsin，美国53711)(Needleman，S.B.Wunsch，C.D.，(1970)，分子生物学杂志(Joumal of Molecular Biology，48，p.443-453))。采用下列设置以GAP进行多肽序列的比对：GAP产生得分为3.0，GAP延伸得分为0.1，本发明中DNA类似序列编码的多肽的成熟部分显示出与SEQ ID NO：2的氨基酸序列的成熟部分优选至少80％，至少90％，更优选至少95％，更优选至少97％，最优选至少99％的同一性。Homology to the parent glucoamylases described above is determined as the degree of identity between two protein sequences that indicates where the first sequence is derived from the second sequence. This homology is suitable for being determined by computer programs known in the art, such as GAP provided by the GCG software package (Wisconsin Software Package Program Manual, 8th Edition, 1994.8, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, U.S. 53711 ) (Needleman, S.B.Wunsch, C.D., (1970), Journal of Molecular Biology (Journal of Molecular Biology, 48, p.443-453)). The comparison of polypeptide sequences is carried out with GAP using the following settings: the GAP generation score is 3.0, the GAP extension score is 0.1, and the mature part of the polypeptide encoded by the DNA similar sequence in the present invention shows the mature part of the amino acid sequence of SEQ ID NO: 2 Preferably at least 80%, at least 90%, more preferably at least 95%, more preferably at least 97%, most preferably at least 99% identity.

在一个实施方案中，亲本葡糖淀粉酶是黑曲霉G1葡糖淀粉酶(Boel等(1984)，EMBO J.3(5)，p.1097-1102(SEQ ID NO：13)。亲本葡糖淀粉酶可以是截短的葡糖淀粉酶，例如，黑曲霉G2葡糖淀粉酶(SEQ ID NO：2)。In one embodiment, the parent glucoamylase is Aspergillus niger G1 glucoamylase (Boel et al. (1984), EMBO J.3(5), p.1097-1102 (SEQ ID NO: 13). Parent glucoamylase The amylase may be a truncated glucoamylase, e.g., Aspergillus niger G2 glucoamylase (SEQ ID NO: 2).

优选地，亲本葡糖淀粉包含SEQ ID NO：2的氨基酸序列；或其等位基因变体；或其具有葡糖淀粉酶活性的片段。Preferably, the parent glucoamylase comprises the amino acid sequence of SEQ ID NO: 2; or its allelic variant; or a fragment thereof with glucoamylase activity.

SEQ ID NO：2的片段是在此氨基酸序列的氨基和/或羧基末端具有一或多个氨基酸缺失的多肽。例如，AMG G2(SEQ ID NO：2)是黑曲霉G1葡糖淀粉酶(Boel等(1984)EMBO J.3(5)，p.1097-1102)的具有葡糖淀粉酶活性的片段。等位基因变体表示占据同一染色体位点的一个基因的两或多个替代形式中的任一种。等位基因变异通过突变自然产生，可能会产生种群内的多态现象。基因突变可以是沉默的(编码的多肽没有改变)或可以编码氨基酸序列已改变的多肽。多肽的等位基因变体是被基因的等位基因变体编码的多肽。Fragments of SEQ ID NO: 2 are polypeptides having one or more amino acid deletions at the amino and/or carboxyl termini of this amino acid sequence. For example, AMG G2 (SEQ ID NO: 2) is a fragment with glucoamylase activity of Aspergillus niger G1 glucoamylase (Boel et al. (1984) EMBO J. 3(5), p. 1097-1102). Allelic variant means any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation and may result in polymorphism within populations. Genetic mutations can be silent (the encoded polypeptide does not change) or can encode a polypeptide whose amino acid sequence has been altered. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.

通过一或多个氨基酸残基的插入或缺失和/或一或多个氨基酸残基被不同氨基酸残基取代，同源的亲本葡糖淀粉酶的氨基酸序列可能与SEQ IDNO：2氨基酸序列不同。优选地，氨基酸改变为较小的性质改变，即并不显著影响蛋白质折叠和/或活性的保守氨基酸的取代；小的缺失，通常为1到约30个氨基酸的缺失；小的氨基-或羧基-末端延伸，如氨基酸末端蛋氨酸残基；最多20-25个残基的小接头肽；或通过改变净电荷或另一基团以利于纯化的小的延伸，如多组氨酸段，抗原表位或结合域。The amino acid sequence of the homologous parent glucoamylase may differ from the amino acid sequence of SEQ ID NO: 2 by insertion or deletion of one or more amino acid residues and/or substitution of one or more amino acid residues by different amino acid residues. Preferably, amino acid changes are minor property changes, i.e., conservative amino acid substitutions that do not significantly affect protein folding and/or activity; small deletions, typically 1 to about 30 amino acid deletions; small amino- or carboxyl - terminal extensions, such as amino acid terminal methionine residues; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another group, such as polyhistidine stretches, antigenic surface bits or binding domains.

在另一实施方案中，分离的亲本葡糖淀粉酶活性被在极低度严谨，优选低度严谨，更优选中度严谨，更优选中高度严谨，进而更优选高度严谨，最优选极高度严谨条件下与核苷酸探针杂交的核酸编码，所述探针在同样的条件下与(i)SEQ ID NO：1的核酸序列，(ii)SEQ ID NO：1的cDNA序列，(iii)(i)或(ii)的亚序列，或(iv)(i)，(ii)或(iii)的互补链杂交(J.Sambrook，E.F.Fritsch，和T.Maniatus，1989，分子克隆，实验室手册，第二版，冷泉港，纽约)。SEQ ID NO：1的亚序列是至少100个核苷酸或优选至少200个核苷酸。而且，亚序列可以编码具有葡糖淀粉酶活性的多肽片段。亲本多肽也可以是具有葡糖淀粉酶活性的多肽的等位基因变体或片段。In another embodiment, the isolated parental glucoamylase activity is measured at very low stringency, preferably low stringency, more preferably moderate stringency, more preferably medium high stringency, even more preferably high stringency, most preferably very high stringency A nucleic acid code that hybridizes with a nucleotide probe under the same conditions, and the probe is under the same conditions with (i) the nucleic acid sequence of SEQ ID NO: 1, (ii) the cDNA sequence of SEQ ID NO: 1, (iii) Subsequences of (i) or (ii), or hybridization of complementary strands of (iv) (i), (ii) or (iii) (J. Sambrook, E.F. Fritsch, and T. Maniatus, 1989, Molecular Cloning, Laboratories Handbook, Second Edition, Cold Spring Harbor, New York). A subsequence of SEQ ID NO: 1 is at least 100 nucleotides or preferably at least 200 nucleotides. Furthermore, a subsequence may encode a fragment of a polypeptide having glucoamylase activity. A parent polypeptide may also be an allelic variant or fragment of a polypeptide having glucoamylase activity.

可采用SEQ ID NO：1的核酸序列或其亚序列，以及SEQ ID NO：2的氨基酸序列或其片段设计核酸探针，以便按照本领域熟知的方法从不同属或种的菌株鉴定和克隆编码具有葡糖淀粉酶活性的多肽的DNA。具体地，这样的探针可用于经标准的Southern印迹方法与目的属或种的基因组DNA或cDNA杂交，以便鉴定并分离本文中相应基因。所述探针可以比完整序列短很多，但长度至少为15，优选至少25，和更优选至少35个核苷酸。也可以使用更长的探针。DNA和RNA探针都可使用。通常标记探针以便检测相应基因(例如，用32P、3H、35S、生物素或抗生物素蛋白标记)。这些探针均包括在本发明中。Nucleic acid probes can be designed using the nucleic acid sequence of SEQ ID NO: 1 or its subsequence, and the amino acid sequence of SEQ ID NO: 2 or a fragment thereof, so as to identify and clone the encoded DNA of a polypeptide having glucoamylase activity. In particular, such probes can be used to hybridize to genomic DNA or cDNA of the genus or species of interest by standard Southern blotting methods in order to identify and isolate the corresponding genes herein. The probes can be much shorter than the complete sequence, but are at least 15, preferably at least 25, and more preferably at least 35 nucleotides in length. Longer probes can also be used. Both DNA and RNA probes can be used. Probes are typically labeled for detection of the corresponding gene (eg, with32P , 3H , 35S , biotin or avidin). These probes are all included in the present invention.

因此，可在从所述其他生物制备的基因组DNA或cDNA文库中筛选与上述探针杂交并编码具有葡糖淀粉酶活性之多肽的DNA。通过琼脂糖或聚丙烯酰胺凝胶电泳或其它分离技术可分离来自所述其他生物的基因组或其他DNA。可将来自所述文库的DNA或分离的DNA转移至并固定在硝酸纤维素或其他适宜的载体材料上。为了鉴定与SEQ ID NO：1或其亚序列同源的克隆或DNA，在Southern印迹中使用该载体材料。为了达到本发明的目的，杂交指在极低至极高严谨条件下，核酸序列与对应于SEQ ID NO：1或其互补链或其亚序列的核酸序列杂交。用X射线胶片检测在这些条件下与所述核酸探针杂交的分子。Accordingly, genomic DNA or cDNA libraries prepared from such other organisms can be screened for DNA that hybridizes to the above probe and encodes a polypeptide having glucoamylase activity. Genomic or other DNA from such other organisms may be separated by agarose or polyacrylamide gel electrophoresis or other separation techniques. DNA from the library or isolated DNA can be transferred to and immobilized on nitrocellulose or other suitable support material. To identify clones or DNA homologous to SEQ ID NO: 1 or a subsequence thereof, the carrier material was used in a Southern blot. For the purposes of the present invention, hybridization refers to the hybridization of a nucleic acid sequence to a nucleic acid sequence corresponding to SEQ ID NO: 1 or its complementary strand or a subsequence thereof under extremely low to extremely high stringency conditions. Molecules that hybridize to the nucleic acid probe under these conditions are detected using X-ray film.

对于至少100个核苷酸的长探针，用2xSSC，0.2％SDS优选至少在45℃(极低严谨度)，更优选50℃(低严谨度)，更优选55℃(中等严谨度)，更优选至少60℃(中等偏高严谨度)，更优选至少65℃(高严谨度)，最优选至少70℃(极高严谨度)将载体材料最后洗涤3次，每次15分钟。For long probes of at least 100 nucleotides, use 2xSSC, 0.2% SDS preferably at least at 45°C (very low stringency), more preferably 50°C (low stringency), more preferably 55°C (medium stringency), More preferably at least 60°C (medium to high stringency), more preferably at least 65°C (high stringency), most preferably at least 70°C (very high stringency) the carrier material is washed 3 times for 15 minutes each.

对于约15个核苷酸至约70个核苷酸的短探针，将严谨度定义为在0.9M NaCl，0.09M Tris-HCl pH7.6，6mM EDTA，0.5％NP-40，1X Denhardt’s溶液，1mM焦磷酸钠，1mM磷酸氢二钠，0.1mM ATP和0.2mg/ml酵母RNA中，在相对于按照Bolton和McCarthy(1962，美国国家科学院进展(Proceedings ofThe National Academy of Sciences USA)48：1390)计算方法计算的Tm低5℃至10℃的温度下，根据标准Southern印迹方法，进行预杂交、杂交和杂交后洗涤。For short probes of about 15 nucleotides to about 70 nucleotides, stringency is defined as a solution in 0.9M NaCl, 0.09M Tris-HCl pH7.6, 6mM EDTA, 0.5% NP-40, 1X Denhardt's solution , 1 mM sodium pyrophosphate, 1 mM disodium hydrogen phosphate, 0.1 mM ATP and 0.2 mg/ml yeast RNA, compared to Bolton and McCarthy (1962, Proceedings of The National Academy of Sciences USA) 48: 1390 ) at a temperature 5°C to 10°C lower than the Tm calculated by the calculation method, according to the standard Southern blotting method, pre-hybridization, hybridization and post-hybridization washing were performed.

对于约15个核苷酸至约70个核苷酸的短探针，在比所计算的Tm低5℃-10℃的温度下，将载体材料用6XSCC加0.1％SDS洗涤一次，15分钟，用6XSSC洗涤两次，每次15分钟。For short probes of about 15 nucleotides to about 70 nucleotides, wash the support material once with 6X SCC plus 0.1% SDS for 15 minutes at a temperature 5°C-10°C lower than the calculated Tm, Wash twice with 6XSSC for 15 minutes each.

本发明还涉及分离的核酸序列，其通过(a)在极低，低，中，中高，高，或极高严谨度下与SEQ ID NO：1或其互补链，或其亚序列杂交；和(b)分离所述核酸序列而产生。亚序列优选至少100个核苷酸的序列，如编码具有葡糖淀粉酶活性的多肽片段的序列。The present invention also relates to isolated nucleic acid sequences that hybridize to SEQ ID NO: 1 or its complementary strand, or a subsequence thereof, by (a) at very low, low, medium, medium high, high, or very high stringency; and (b) produced by isolating said nucleic acid sequence. The subsequence is preferably a sequence of at least 100 nucleotides, such as a sequence encoding a polypeptide fragment having glucoamylase activity.

所涉及的亲本葡糖淀粉酶具有SEQ ID NO：2的成熟葡糖淀粉酶的葡糖淀粉酶活性的至少20％，优选至少40％，更优选60％，更优选80％，更优选90％，最优选100％。The parent glucoamylase involved has at least 20%, preferably at least 40%, more preferably 60%, more preferably 80%, more preferably 90% of the glucoamylase activity of the mature glucoamylase of SEQ ID NO: 2 , most preferably 100%.

克隆能编码亲本葡糖淀粉酶的DNA序列Cloning of the DNA sequence encoding the parental glucoamylase

用本领域熟知的各种方法，从生产所述葡糖淀粉酶的任何细胞或微生物中可分离编码亲本葡糖淀粉酶的DNA序列。首先应用来自产所述葡糖淀粉酶的生物体的染色体DNA或信使RNA构建基因组DNA和/或cDNA文库。然后，如果所述葡糖淀粉酶的氨基酸序列是已知的，则可合成标记的寡核苷酸探针并用于从制备自目标生物的基因组文库中鉴定编码葡糖淀粉酶的克隆。或者，可用含有与另一种已知葡糖淀粉酶基因同源之序列的标记型寡核苷酸探针作为探针，利用极低到极高严谨度的杂交和洗涤条件鉴定编码葡糖淀粉酶的克隆。如上述。The DNA sequence encoding the parent glucoamylase can be isolated from any cell or microorganism that produces the glucoamylase by various methods well known in the art. Genomic DNA and/or cDNA libraries are first constructed using chromosomal DNA or messenger RNA from the glucoamylase-producing organism. Then, if the amino acid sequence of the glucoamylase is known, labeled oligonucleotide probes can be synthesized and used to identify glucoamylase-encoding clones from genomic libraries prepared from the target organism. Alternatively, a labeled oligonucleotide probe containing sequence homology to another known glucoamylase gene can be used as a probe to identify genes encoding glucoamylase using very low to very high stringency hybridization and wash conditions. Enzyme cloning. As above.

用于鉴定葡糖淀粉酶编码克隆的另一种方法涉及将基因组DNA的片段插入表达载体，例如质粒中，用所得基因组DNA文库转化葡糖淀粉酶阴性细菌，然后将转化的细菌铺板在含葡糖淀粉酶底物(即麦芽糖)的琼脂上，由此鉴定表达葡糖淀粉酶的克隆。Another method for identifying glucoamylase-encoding clones involves inserting fragments of genomic DNA into expression vectors, such as plasmids, transforming glucoamylase-negative bacteria with the resulting genomic DNA library, and then plating the transformed bacteria on glucoamylase-containing Glucoamylase-expressing clones were identified on the agar of the glucoamylase substrate (ie, maltose).

或者，通过现有标准方法，例如S.L.Beaucage和M.H.Caruthers所述的亚磷酰胺法(1981)，Tetrahedron Letters 22，p.1859-1869或者Matthes等(1984)，EMBO J.3，p.801-805所述的方法合成编码所述酶的DNA序列。在亚磷酰胺方法中，用例如自动DNA合成仪合成寡核苷酸，使其纯化、退火、连接，然后克隆到适宜的载体中。Alternatively, by existing standard methods, such as the phosphoramidite method described by S.L. Beaucage and M.H. Caruthers (1981), Tetrahedron Letters 22, p.1859-1869 or Matthes et al. (1984), EMBO J.3, p.801- The method described in 805 synthesizes the DNA sequence encoding the enzyme. In the phosphoramidite method, oligonucleotides are synthesized using, for example, an automatic DNA synthesizer, purified, annealed, ligated, and cloned into a suitable vector.

最后，所述DNA序列可以是按照标准技术通过连接合成的、基因组的或cDNA来源的序列(根据需要，对应于完整DNA序列各部分的片段)而制备的基因组序列与合成序列的混合序列、合成序列与cDNA序列的混合序列或者基因组序列与cDNA的混合序列。还可通过聚合酶链反应(PCR)，用特异性引物，按US4683202或R.K.Saiki等(1988)，科学239，1988，pp.487-491所述制备DNA序列。Finally, the DNA sequence may be a mixed sequence of genomic and synthetic sequences prepared according to standard techniques by ligating sequences of synthetic, genomic or cDNA origin (fragments corresponding to parts of the complete DNA sequence, as required), synthetic A mixed sequence of sequence and cDNA sequence or a mixed sequence of genomic sequence and cDNA. DNA sequences can also be prepared by polymerase chain reaction (PCR) using specific primers as described in US4683202 or R.K. Saiki et al. (1988), Science 239, 1988, pp. 487-491.

定点诱变site-directed mutagenesis

分离了编码葡糖淀粉酶的DNA序列并鉴定了合乎要求的突变位点后，用合成的寡核苷酸导入突变。这些寡核苷酸含有位于所需突变位点侧的核苷酸序列；在寡核苷酸合成的过程中插入突变核苷酸。在具体的方法中，在携带葡糖淀粉酶基因的载体中产生单链DNA缺口，编码葡糖淀粉酶的序列。然后，将携带所需突变的合成核苷酸与单链DNA的同源部分退火。用DNA聚合酶I(Klenow片段)填平剩余缺口，用T4连接酶连接构建体。Morinaga等(1984)，生物技术2，p.646-639描述了该方法的具体实施例。US4760025公开了通过对表达盒进行微小改变来导入编码多个突变的寡核苷酸。但是，通过Morinaga方法可以在任何时间导入更多种的突变，因为可以导入不同长度的众多寡核苷酸。After isolation of the DNA sequence encoding the glucoamylase and identification of desirable mutation sites, mutations are introduced using synthetic oligonucleotides. These oligonucleotides contain nucleotide sequences flanking the desired mutation site; the mutant nucleotides are inserted during oligonucleotide synthesis. In a specific method, a single-stranded DNA gap, encoding the glucoamylase sequence, is created in a vector carrying the glucoamylase gene. Synthetic nucleotides carrying the desired mutation are then annealed to the homologous portion of the single-stranded DNA. The remaining gaps were filled with DNA polymerase I (Klenow fragment), and the constructs were ligated with T4 ligase. Specific examples of this method are described by Morinaga et al. (1984), Biotechnology 2, p. 646-639. US4760025 discloses the introduction of oligonucleotides encoding multiple mutations by making minor changes to the expression cassette. However, a greater variety of mutations can be introduced at any one time by the Morinaga method because numerous oligonucleotides of different lengths can be introduced.

Nelson和Long(1989)，分析生物化学(Analytical Biochemistry)180，p.147-151描述了另一种将突变导入编码葡糖淀粉酶的DNA序列的方法。该方法包括经3步产生PCR片段，该片段含有通过用化学合成的DNA链作为PCR反应中的一个引物而导入的所需突变。通过用限制核酸内切酶裂解从PCR-产生的片段中分离携带突变的DNA片段，然后再插入到表达质粒中。Nelson and Long (1989), Analytical Biochemistry 180, p. 147-151 describe another method of introducing mutations into the DNA sequence encoding glucoamylases. The method involves three steps to generate a PCR fragment containing the desired mutation introduced by using a chemically synthesized DNA strand as a primer in a PCR reaction. The mutation-carrying DNA fragment was isolated from the PCR-generated fragment by cleavage with restriction endonucleases, and then inserted into the expression plasmid.

此外，Sierks等(1989)“在泡盛曲霉葡糖淀粉酶的活性位点Trp120的定点诱变”，蛋白质工程(Protein Eng.)，2，621-625；Sierks等(1990)“通过在活性位点Asp176，Glu179，Glu180进行定点诱变来确定泡盛曲霉葡糖淀粉酶的催化机理”蛋白质工程，32，193-198；还记述了曲霉属葡糖淀粉酶中的定点诱变。In addition, Sierks et al. (1989) "Site-directed mutagenesis at the active site Trp120 of Aspergillus awamori glucoamylase", Protein Engineering (Protein Eng.), 2, 621-625; Sierks et al. Site-directed mutagenesis of Asp176, Glu179, Glu180 to determine the catalytic mechanism of Aspergillus awamori glucoamylase" Protein Engineering, 32, 193-198; also describes site-directed mutagenesis in Aspergillus glucoamylase.

定域随机诱变localized random mutagenesis

随机诱变可有利地定域于所述亲代葡糖淀粉酶的一部分。例如，当酶的某些区域被鉴定为对酶的给定特性特别重要时，以及当预期修饰能导致具有改良特性的变体时，定域随机诱变较为有利。当亲代酶的三级结构已被阐明，且所述结构与酶的功能相关时，一般可鉴定出所述区域。Random mutagenesis may advantageously be localized to a portion of the parent glucoamylase. For example, localized random mutagenesis is advantageous when certain regions of an enzyme are identified as being particularly important for a given property of the enzyme, and when modifications are expected to result in variants with improved properties. Such regions are generally identified when the tertiary structure of the parent enzyme has been elucidated and the structure is related to the function of the enzyme.

通过使用上述的PCR产生的诱变技术或本领域已知的任何其它适当的技术，可以方便地进行定域的或区域-特异性的随机诱变。或者，通过例如插入适当的载体来分离编码待修饰的DNA序列部分的DNA序列，随后可使用上述任何诱变方法对所述部分进行诱变。Localized or region-specific random mutagenesis may be conveniently performed using the PCR-generated mutagenesis technique described above, or any other suitable technique known in the art. Alternatively, the DNA sequence encoding the portion of the DNA sequence to be modified is isolated, for example by insertion into an appropriate vector, and said portion can subsequently be mutagenized using any of the mutagenesis methods described above.

提供本发明变体的备选方法包括基因改组，例如WO 95/22625(来自Affymax Technologies N.V.)或WO 96/00343(来自Novo Nordisk A/S)中所述。Alternative methods of providing variants of the invention include gene shuffling, such as described in WO 95/22625 (from Affymax Technologies N.V.) or WO 96/00343 (from Novo Nordisk A/S).

葡糖淀粉酶变体的表达Expression of glucoamylase variants

根据本发明，可使用表达载体，以酶的形式表达通过上述方法，或通过本领域已知的任何其它方法产生的编码变体的DNA序列，所述表达载体一般包括编码启动子，操纵子，核糖体结合位点，翻译起始信号的控制序列，并任选包括阻抑基因或多种激活基因。According to the present invention, expression vectors can be used to express in the form of enzymes the DNA sequences encoding variants produced by the above methods, or by any other methods known in the art. Said expression vectors generally include encoding promoters, operators, Ribosome binding sites, control sequences for translation initiation signals, and optionally include repressor genes or various activator genes.

表达载体Expression vector

带有编码本发明葡糖淀粉酶变体DNA序列的重组表达载体是任何可以方便地进行重组DNA方法的载体，载体的选择取决于将被引入的宿主细胞。载体可以是，当被引入宿主细胞时可整合进宿主细胞基因组并与其所整合进的染色体一起复制的那些。合适的表达载体例子包括pMT838。The recombinant expression vector carrying the DNA sequence encoding the glucoamylase variant of the present invention is any vector that can conveniently carry out recombinant DNA methods, and the choice of the vector depends on the host cell to be introduced. Vectors may be those which, when introduced into a host cell, integrate into the host cell genome and replicate with the chromosome into which they have been integrated. Examples of suitable expression vectors include pMT838.

启动子Promoter

载体中，DNA序列应该可操作连接适合的启动子序列。启动子可以是在所选宿主细胞中表现出转录活性的任何DNA序列，可以从编码对宿主细胞同源或异源的蛋白的基因获得。In the vector, the DNA sequence should be operably linked to a suitable promoter sequence. The promoter may be any DNA sequence that exhibits transcriptional activity in the host cell of choice, and may be obtained from genes encoding proteins either homologous or heterologous to the host cell.

引导编码本发明葡糖淀粉酶变体的DNA序列转录，特别是在细菌宿主中，转录的适合的启动子是例如，大肠杆菌lac操纵子，天蓝色链霉菌琼脂酶基因dagA，地衣芽孢杆菌α-淀粉酶基因(amyL)、嗜热脂肪芽孢杆菌产麦芽淀粉酶基因(amyM)、解淀粉芽孢杆菌α-淀粉酶基因(amyQ)、枯草芽孢杆菌xy1A和xy1B基因等的启动子。对于在真菌宿主中进行转录，可用的启动子的例子有来自米曲霉TAKA淀粉酶基因，酿酒酵母TPI(丙糖磷酸异构酶)(Alber等(1982)，J.Mol.Appl.Genet1，p.419-434，米赫根毛霉(Rhizomucormiehei)天冬氨酸蛋白酶，黑曲霉中性α-淀粉酶，黑曲霉酸稳定的α-淀粉酶，黑曲霉葡糖淀粉酶，米赫根毛霉脂肪酶，米曲霉碱性蛋白酶，米曲霉丙糖磷酸异构酶或构巢曲霉乙酰胺酶的启动子。To direct the transcription of the DNA sequence encoding the glucoamylase variant of the present invention, particularly in bacterial hosts, suitable promoters for transcription are, for example, the Escherichia coli lac operon, Streptomyces coelicolor agarase gene dagA, Bacillus licheniformis alpha - promoters of the amylase gene (amyL), the Bacillus stearothermophilus malting amylase gene (amyM), the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), the Bacillus subtilis xy1A and xy1B genes, etc. For transcription in fungal hosts, examples of usable promoters are the TAKA amylase gene from Aspergillus oryzae, the TPI (triose phosphate isomerase) from Saccharomyces cerevisiae (Alber et al. (1982), J. Mol. Appl. Genet 1, p .419-434, Rhizomucor miehei (Rhizomucormiehei) aspartic protease, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid-stabilized alpha-amylase, Aspergillus niger glucoamylase, Rhizomucor miehei lipase , the promoter of Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase or Aspergillus nidulans acetamidase.

表达载体Expression vector

本发明表达载体也含有适当的转录终止子，在真核生物中，还含有与编码本发明α-淀粉酶变体的DNA序列可操作相连的聚-腺苷酸化序列。终止和聚腺苷酸化序列可适当地得自与启动子相同的来源。The expression vector of the present invention also contains an appropriate transcription terminator and, in eukaryotes, a poly-adenylation sequence operably linked to the DNA sequence encoding the α-amylase variant of the present invention. Termination and polyadenylation sequences may suitably be obtained from the same sources as the promoter.

载体可进一步含有能使载体在所述宿主细胞中复制的DNA序列。所述序列的例子是质粒pUC19，pACYC177，pUB110，pE194，pAMB1和pIJ702的复制起点。A vector may further contain DNA sequences that enable the vector to replicate in said host cell. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUB110, pE194, pAMB1 and pIJ702.

载体也可含有选择标记，例如其产物可以补偿宿主细胞缺陷的基因，如枯草芽孢杆菌或地衣芽孢杆菌的dal基因，或能赋予抗生素抗性的基因，所述抗生素抗性如氨苄青霉素，卡那霉素，氯霉素或四环素抗性。另外，载体可含有曲霉属选择标记，如amdS，argB，niaD和sC，产生潮霉素抗性的标记，或者可通过WO91/17243中所述的共-转化来完成选择。The vector may also contain a selectable marker, such as a gene whose product compensates for a defect in the host cell, such as the dal gene of Bacillus subtilis or Bacillus licheniformis, or a gene that confers resistance to antibiotics such as ampicillin, kana Resistance to chloramphenicol, chloramphenicol, or tetracycline. Alternatively, the vector may contain Aspergillus selection markers, such as amdS, argB, niaD and sC, markers conferring hygromycin resistance, or selection may be accomplished by co-transformation as described in WO 91/17243.

分别连接编码葡糖淀粉酶变体的本发明DNA构建体，启动子，终止子和其它元件，并将它们插入含有复制所需信息的适当载体的方法是本领域技术人员众所周知的(参见例如Sambrook等，分子克隆：实验室手册，第2版，冷泉港，1989)。Methods for ligating the DNA constructs of the present invention encoding glucoamylase variants, promoters, terminators and other elements, respectively, and inserting them into appropriate vectors containing the information required for replication are well known to those skilled in the art (see e.g. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, 1989).

宿主细胞host cell

含有本发明的上述DNA构建体或表达载体的本发明的细胞，在本发明葡糖淀粉酶变体的重组生产中作为宿主细胞较有利。可以方便地通过将本发明的DNA构建体整合进宿主染色体，从而用该DNA构建体转化该细胞。由于所述DNA序列更可能在细胞中保持稳定，此整合通常被认为是有利的。DNA构建体整合进入宿主染色体可按照常规方法，例如，同源或异源重组来进行。备选地，可用上述与不同类型宿主细胞有关的表达载体转化细胞。The cells of the invention containing the aforementioned DNA constructs or expression vectors of the invention are advantageous as host cells in the recombinant production of the glucoamylase variants of the invention. The cell is conveniently transformed with the DNA construct of the invention by integrating the DNA construct into the host chromosome. This integration is generally considered to be advantageous since the DNA sequence is more likely to remain stable in the cell. Integration of the DNA construct into the host chromosome can be performed according to conventional methods, eg, homologous or heterologous recombination. Alternatively, cells can be transformed with the expression vectors described above in relation to different types of host cells.

本发明的细胞可以是较高等生物的细胞，如哺乳动物或昆虫的细胞，但优选是微生物细胞，如细菌或真菌(包括酵母)的细胞。The cells of the invention may be cells of higher organisms, such as mammalian or insect cells, but are preferably microbial cells, such as bacterial or fungal (including yeast) cells.

适当细菌的例子如下：革兰氏阳性细菌，如枯草芽孢杆菌，地衣芽孢杆菌，迟缓芽孢杆菌，短芽孢杆菌，嗜热脂肪芽孢杆菌，嗜碱芽孢杆菌，解淀粉芽孢杆菌，凝结芽孢杆菌，环状芽孢杆菌，灿烂芽孢杆菌，巨大芽孢杆菌，苏云金芽孢杆菌，浅青紫链霉菌或鼠灰链霉菌；或革兰氏阴性细菌，如大肠杆菌。细菌的转化可以，例如，通过原生质体转化或使用感受态细胞以其本身已知的方式进行。Examples of suitable bacteria are as follows: Gram-positive bacteria such as Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus spirochete, Bacillus cannula, Bacillus megaterium, Bacillus thuringiensis, Streptomyces lividans, or Streptomyces grinotis; or Gram-negative bacteria such as Escherichia coli. Transformation of bacteria can be performed, for example, by protoplast transformation or using competent cells in a manner known per se.

酵母优选选自糖酵母属或裂殖糖酵母属，例如酿酒酵母。The yeast is preferably selected from the genus Saccharomyces or Schizosaccharomyces, eg Saccharomyces cerevisiae.

宿主细胞可以是丝状真菌，例如曲霉属某个种的菌株，最优选米曲霉或黑曲霉，或镰孢属菌株，如尖孢镰孢(Fusarium oxysporium)，禾谷镰孢(完整状态即Gribberella zeae，以前为Sphaeria zeae，又名Gibberella roseum和Gibberella roseum f.sp.cerealis)，或硫色镰孢(完整状态即Gribberella puricaris，又名三隔镰孢，杆孢状镰孢，接骨木镰孢，玫瑰色镰孢，玫瑰色镰孢的禾谷镰孢变种)，Fusarium cerealis(又名Fusarium crookwellense)，或Fusariumvenenatum。The host cell may be a filamentous fungus, such as a strain of Aspergillus spp., most preferably Aspergillus oryzae or Aspergillus niger, or a strain of Fusarium, such as Fusarium oxysporium, Fusarium graminearum (full state i.e. Gribberella zeae, formerly Sphaeria zeae, also known as Gibberella roseum and Gibberella roseum f.sp.cerealis), or Fusarium sulfur (full state is Gribberella puricaris, also known as Fusarium three septa, Fusarium baculum, Fusarium elder , Fusarium rosea, Fusarium graminearum var. rosea, Fusarium cerealis (aka Fusarium crookwellense), or Fusarium venenatum.

在本发明优选实施方案中宿主细胞是蛋白酶缺陷型菌株或蛋白酶阴性菌株。In a preferred embodiment of the invention the host cell is a protease deficient strain or a protease negative strain.

例如碱性蛋白酶基因“alp”缺失的米曲霉JaL125就是蛋白酶缺陷型菌株。在WO 97/35956(Novo Nordisk)，或欧洲专利429,490中有此菌株的描述。For example, Aspergillus oryzae JaL125 with the deletion of the alkaline protease gene "alp" is a protease-deficient strain. This strain is described in WO 97/35956 (Novo Nordisk), or European Patent 429,490.

丝状真菌细胞可通过涉及原生质体形成和原生质体转化及随后使细胞壁以其本身的方式再生的一种方法来转化。在EP238023(Novo Nordisk A/S)中描述了用曲霉作宿主微生物，其内容在此引作参考。Filamentous fungal cells can be transformed by a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall in its own right. The use of Aspergillus as a host microorganism is described in EP238023 (Novo Nordisk A/S), the contents of which are incorporated herein by reference.

生产葡糖淀粉酶变体的方法Methods of producing glucoamylase variants

在更进一步的方面，本发明涉及生产本发明葡糖淀粉酶变体的方法，该方法包括在利于生产变体并从细胞及/或培养液中回收该变体的条件下培养宿主细胞。In a still further aspect, the invention relates to a method of producing a glucoamylase variant of the invention comprising culturing a host cell under conditions favorable for production of the variant and recovery of the variant from the cell and/or culture medium.

用于培养细胞的培养基可以是适于目的宿主细胞生长并表达本发明葡糖淀粉酶变体的任何常规培养基。可用的培养基可从销售商购得或按照已公开的配方(例如按美国典型培养物保藏中心的目录中所述)制备。The medium used for culturing the cells may be any conventional medium suitable for the growth of the intended host cell and expressing the glucoamylase variant of the present invention. Useful media are available from commercial suppliers or may be prepared according to published recipes (eg, as described in catalogs of the American Type Culture Collection).

通过已熟知的方法从培养液中可以方便地回收宿主细胞分泌的葡糖淀粉酶变体，所述方法包括通过离心或过滤从培养液分离细胞，通过盐如硫酸铵沉淀培养基质中的蛋白类成分，然后使用层析的方法，如离子交换层析，亲和层析，等。Glucoamylase variants secreted by host cells are conveniently recovered from the culture medium by well-known methods, including separation of the cells from the culture medium by centrifugation or filtration, precipitation of proteins from the culture medium by salts such as ammonium sulfate components, and then use chromatographic methods such as ion exchange chromatography, affinity chromatography, etc.

淀粉转化starch conversion

本发明提供一种利用本发明的葡糖淀粉酶变体从淀粉生产葡萄糖及类似物的方法。通常，该方法包括在α-淀粉酶存在下部分水解前体淀粉，然后在葡糖淀粉酶存在下通过切割α-(1→4)和α-(1→6)糖苷键从淀粉或相关寡糖和多糖分子的非还原末端进一步水解释放D-葡萄糖。The invention provides a method for producing glucose and the like from starch using the glucoamylase variants of the invention. Typically, the method involves partial hydrolysis of precursor starch in the presence of α-amylase, followed by cleavage of α-(1→4) and α-(1→6) glycosidic linkages in the presence of glucoamylase from starch or related oligosaccharides. The non-reducing ends of sugar and polysaccharide molecules are further hydrolyzed to release D-glucose.

利用α-淀粉酶进行的前体淀粉的部分水解使淀粉分子通过水解内部的α-(1→4)键而初步分解。在商业应用中，利用α-淀粉酶的初步水解在约105℃下进行。所处理的淀粉浓度很高，通常30％-40％固含量。在这种提高的温度下的初步水解一般进行5分钟。然后将部分水解的淀粉转移到第二个罐，85-90℃温育约1小时，得到10-15的葡萄糖当量值(D.E.)。Partial hydrolysis of the precursor starch by alpha-amylases initially breaks down the starch molecules by hydrolyzing the internal alpha-(1→4) linkages. In commercial applications, primary hydrolysis with alpha-amylase is performed at about 105°C. The processed starch concentration is very high, usually 30%-40% solid content. Preliminary hydrolysis at this elevated temperature is generally carried out for 5 minutes. The partially hydrolyzed starch was then transferred to a second tank and incubated at 85-90°C for about 1 hour to obtain a dextrose equivalent (D.E.) of 10-15.

通常在一个单独的罐中，在降低至30-60℃的温度下，在有葡糖淀粉酶时进一步水解从淀粉或相关寡糖和多糖分子的非还原末端释放出的D-葡萄糖。该底物液体的温度优选降至55-60℃之间。该溶液的pH值从6-6.5降至3-5.5。优选pH为4-4.5。将葡糖淀粉酶加入该溶液中，使反应进行24-72小时，优选36-48小时。D-glucose released from the non-reducing ends of starch or related oligo- and polysaccharide molecules is further hydrolyzed in the presence of glucoamylase, usually in a separate tank at reduced temperatures to 30-60°C. The temperature of the substrate liquid is preferably reduced to between 55-60°C. The pH of the solution dropped from 6-6.5 to 3-5.5. A pH of 4-4.5 is preferred. Glucoamylase is added to the solution and the reaction is allowed to proceed for 24-72 hours, preferably 36-48 hours.

通过使用本发明的热稳定的葡糖淀粉酶变体可以在比传统分批糖化过程更高的温度下进行糖化过程。依照本发明，糖化可在60-80℃以上进行，优选63-75℃。其既可用于传统分批方法(如上所述)也可用于连续糖化。By using the thermostable glucoamylase variants of the invention it is possible to carry out the saccharification process at higher temperatures than traditional batch saccharification processes. According to the present invention, saccharification may be carried out above 60-80°C, preferably 63-75°C. It can be used in both traditional batch processes (as described above) and continuous saccharification.

实际上，包括一或多步膜分离步骤，即过滤步骤的连续糖化过程必须在60℃以上进行以保持一个合理的高膜通量。因此，本发明热稳定的葡糖淀粉酶变体为在工业化糖化过程可接受的合理价格和时间内进行大规模连续糖化处理提供了可能。依照本发明甚至可以缩短糖化时间。In practice, continuous saccharification processes involving one or more membrane separation steps, ie filtration steps, must be performed above 60°C to maintain a reasonably high membrane flux. Therefore, the thermostable glucoamylase variants of the present invention provide the possibility for large-scale continuous saccharification at a reasonable price and time acceptable to industrial saccharification processes. According to the invention it is even possible to shorten the mashing time.

本发明葡糖淀粉酶变体(如AMG变体)的活性在60-80℃比在传统采用的温度范围30-60℃时通常要高很多。因此，当所述葡糖淀粉酶实施糖化过程时通过提高温度可以缩短时间。The activity of the glucoamylase variants (eg AMG variants) of the invention is generally much higher at 60-80°C than at the traditionally employed temperature range of 30-60°C. Therefore, the time can be shortened by increasing the temperature when the glucoamylase performs the saccharification process.

此外，提高热稳定性改善了T1/2(半衰期，如“材料和方法”部分所定义)。由于本发明葡糖淀粉酶变体改进了热稳定性，所以糖化过程中仅需要添加小量葡糖淀粉酶以替代已失活的酶。依照本发明，更多的葡糖淀粉酶在糖化过程中保持活性。此外，在63℃以上进行糖化可以减少微生物污染的风险。In addition, increasing thermal stability improves T 1/2 (half-life, as defined in the "Materials and Methods" section). Due to the improved thermostability of the glucoamylase variants of the present invention, only a small amount of glucoamylase needs to be added to replace the inactivated enzyme during saccharification. According to the invention, more glucoamylases remain active during saccharification. In addition, performing saccharification above 63 °C reduces the risk of microbial contamination.

在JP 3-224493；JP1-191693；JP62-272987；EP452,238中记述了使用本发明葡糖淀粉酶变体进行糖化过程的实例。Examples of saccharification processes using the glucoamylase variants of the present invention are described in JP 3-224493; JP1-191693; JP62-272987; EP452,238.

在本发明方法中，本发明葡糖淀粉酶变体可与一种能水解至少有4个糖基的分子中的α-(1→6)糖苷键的酶联合使用。优选地，本发明葡糖淀粉酶变体可与支链淀粉酶或异淀粉酶联合使用。在G.M.A.van Beynum等，淀粉转化技术，Marcel Dekker，New York，1985，101-142中提出了支链淀粉酶和异淀粉酶的去支链应用，所述酶的分子性质，所述酶和葡糖淀粉酶的潜在用途。In the method of the present invention, the glucoamylase variant of the present invention can be used in combination with an enzyme capable of hydrolyzing α-(1→6) glycosidic bonds in molecules having at least 4 glycosyl groups. Preferably, the glucoamylase variants of the invention may be used in combination with pullulanase or isoamylase. The debranching application of pullulanase and isoamylase, the molecular properties of the enzyme, the enzyme and glucose Potential uses of glycoamylases.

本发明的又一方面涉及本发明葡糖淀粉酶变体在淀粉转化过程中的应用。A further aspect of the invention relates to the use of the glucoamylase variants of the invention in starch conversion processes.

此外，本发明葡糖淀粉酶变体可用于包括连续糖化步骤的连续淀粉转化过程。Furthermore, the glucoamylase variants of the invention may be used in continuous starch conversion processes comprising successive saccharification steps.

本发明葡糖淀粉酶变体也可以以固定化形式使用。这适于并经常用于生产麦芽糖糊精或葡萄糖浆，或特定的糖浆，如麦芽糖糖浆，还可用于与果糖浆生产有关的寡糖提余液(raffinate)流。The glucoamylase variants of the invention may also be used in immobilized form. This is suitable and often used for the production of maltodextrin or glucose syrup, or specific syrups such as maltose syrup, and also for the oligosaccharide raffinate stream associated with the production of fructose syrup.

依照本发明，本发明的AMG变体也可用于生产醇类，例如燃用或食用醇类。US 5231017记述了其中一个方法。材料和方法酶：According to the invention, the AMG variants of the invention can also be used in the production of alcohols, such as fuel or edible alcohols. One of these methods is described in US 5231017. Materials and Methods Enzymes:

AMG G1：公开在Boel等，(1984)，EMBO J.3(5)，1097-1102，(SEQ IDNO：13)中的黑曲霉葡糖淀粉酶G1，可从Novo Nordisk得到。AMG G1: Aspergillus niger glucoamylase G1 disclosed in Boel et al., (1984), EMBO J.3(5), 1097-1102, (SEQ ID NO: 13), available from Novo Nordisk.

AMG G2：截短的黑曲霉葡糖淀粉酶G1，表示为SEQ ID NO：2，可从Novo Nordisk得到。AMG G2: truncated Aspergillus niger glucoamylase G1, represented as SEQ ID NO: 2, available from Novo Nordisk.

溶液：Solution:

缓冲液：0.05M乙酸钠(1L milli-Q-water中6.8g)，pH4.5Buffer: 0.05M sodium acetate (6.8g in 1L milli-Q-water), pH4.5

终止液：0.4M NaOHStop solution: 0.4M NaOH

GOD-perid，124036，Boehringer MannheimGOD-perid, 124036, Boehringer Mannheim

底物：Substrate:

麦芽糖：29mM(100ml 50mM乙酸钠中1g麦芽糖，pH4.5)(Sigma)Maltose: 29mM (1g maltose in 100ml 50mM sodium acetate, pH4.5) (Sigma)

麦芽庚糖：10mM，115mg/10ml(Sigma)Maltoheptose: 10mM, 115mg/10ml (Sigma)

宿主细胞：Host cell:

米曲霉JaL125：米曲霉IFO4177，可从发酵研究所，Osaka；17-25 JusoHammachi 2-Chome Yodogawa-ku，Osaka，日本得到，其采用米曲霉pyrG基因作标记，并通过一步基因取代方法(G.May，“丝状真菌的应用分子遗传学(Applied Molecular Genetics of Filamentous Fungi”(1982)，p.1-25.Eds.J.R.Knghorn和G.Turner；Blackie Academic and Professional)使碱性蛋白酶基因“alp”缺失(Murakami K等(1991)，Agric.Biol.Chem.55，p.2807-2811)。WO 97/35956(Novo Nordisk)中进一步公开了菌株JaL125。Aspergillus oryzae JaL125: Aspergillus oryzae IFO4177 can be obtained from the Institute of Fermentation, Osaka; 17-25 JusoHammachi 2-Chome Yodogawa-ku, Osaka, Japan, which uses the Aspergillus oryzae pyrG gene as a marker, and through a one-step gene replacement method (G. May, "Applied Molecular Genetics of Filamentous Fungi" (1982), p.1-25. Eds. J.R. Knghorn and G. Turner; Blackie Academic and Professional) makes the alkaline protease gene "alp " deletion (Murakami K et al. (1991), Agric. Biol. Chem. 55, p. 2807-2811). Strain JaL125 is further disclosed in WO 97/35956 (Novo Nordisk).

微生物：microorganism:

菌株：酿酒酵母(S.cerevisiae)YNG318：MATαleu2-Δ2 ura3-52 his4-539 pep4-Δ1[cir+]Strain: Saccharomyces cerevisiae (S.cerevisiae) YNG318: MATαleu2-Δ2 ura3-52 his4-539 pep4-Δ1[cir+]

质粒：Plasmid:

pCAMG91：参见图1，包含黑曲霉G1葡糖淀粉酶(AMG G1)的质粒。Boel等(1984)，EMBO J.3(7)，p1581-1585中记述了pCAMG91的构建。pCAMG91: See Figure 1, plasmid containing Aspergillus niger G1 glucoamylase (AMG G1 ). The construction of pCAMG91 is described in Boel et al. (1984), EMBO J.3(7), p1581-1585.

pMT838：编码截短的黑曲霉葡糖淀粉酶G2(SEQ ID NO：2)的质粒pMT838: Plasmid encoding truncated Aspergillus niger glucoamylase G2 (SEQ ID NO: 2)

pJSO026：(酿酒酵母表达质粒)(J.S.Okkels，(1996)“pYES载体中URA3-启动子的缺失提高了酿酒酵母中真菌脂肪酶的表达水平。DNA重组生物技术III：生物学和工程学的整合，vol.782，Annals of the New York Academy ofSciences”)。更特定地，通过用来自酿酒酵母的组成型表达的TPI(丙糖磷酸异构酶)启动子(Albert和karwasaki，(1982)，J.Mol.Appl.Genet.，1，419-434)，取代pYES2.0的诱导型GAL1-启动子，并缺失URA3启动子的一部分，可从pYES2.0得到表达质粒pJSO37。pJSO026: (Saccharomyces cerevisiae expression plasmid) (J.S. Okkels, (1996) "Deletion of the URA3-promoter in the pYES vector increases expression levels of fungal lipase in Saccharomyces cerevisiae. Recombinant DNA Biotechnology III: Integrating Biology and Engineering , vol.782, Annals of the New York Academy of Sciences"). More specifically, by using the constitutively expressed TPI (triose phosphate isomerase) promoter from Saccharomyces cerevisiae (Albert and karwasaki, (1982), J. Mol. Appl. Genet., 1, 419-434), The expression plasmid pJSO37 was obtained from pYES2.0 by replacing the inducible GAL1-promoter of pYES2.0 and deleting part of the URA3 promoter.

方法：method:

酿酒酵母YNG318的转化Transformation of Saccharomyces cerevisiae YNG318

将DNA片段和开放型载体混合并通过标准方法转化进入酿酒酵母YNG318。The DNA fragment and open vector were mixed and transformed into S. cerevisiae YNG318 by standard methods.

测定比活并表示为kcat(sec.-1)Specific activity was determined and expressed as k cat (sec. -1 )

750μL底物(1％麦芽糖，50mM乙酸钠，pH4.3)在所选温度例如37℃或60 ℃温育5分钟。750 μL of substrate (1% maltose, 50 mM sodium acetate, pH 4.3) was incubated for 5 minutes at the selected temperature, eg 37°C or 60°C.

加入50μL稀释在乙酸钠中的酶。分别在0、3、6、9、12分钟后取100μL等份试样，转移到100μL0.4M氢氧化钠中终止反应。空白也包括在内。Add 50 µL of enzyme diluted in sodium acetate. Aliquots of 100 μL were taken after 0, 3, 6, 9, and 12 minutes and transferred to 100 μL of 0.4M NaOH to terminate the reaction. Blanks are also included.

将20μL移到微滴板上，加入200μL GOD-Perid溶液。室温下温育30分钟后650nm测定吸收值。Pipette 20 μL to a microtiter plate and add 200 μL of GOD-Perid solution. Absorbance was measured at 650 nm after incubation at room temperature for 30 minutes.

用葡萄糖作为标准，比活计算为kcat(sec.-1)。Using glucose as a standard, the specific activity was calculated as k cat (sec. -1 ).

测定AGU活性并表示为AGU/mgAGU activity was determined and expressed as AGU/mg

一个Novo淀粉葡糖苷酶单位(AGU)定义为37℃，pH4.3下每分钟水解1微摩尔麦芽糖的酶量。此分析方法的详述(AEL-SM-0131)可要求NovoNordisk提供。One Novo amyloglucosidase unit (AGU) is defined as the amount of enzyme that hydrolyzes 1 micromole of maltose per minute at 37°C and pH 4.3. A detailed description of this analytical method (AEL-SM-0131) is available from NovoNordisk upon request.

用Boehringer Mannheim，124036的葡萄糖GOD-Perid试剂盒以修饰的(AEL-SM-0131)方法将活性测定为AGU/ml。标准品：AMG-标准品，批次7-1195，195AGU/ml。The activity was determined as AGU/ml using the glucose GOD-Perid kit from Boehringer Mannheim, 124036 with a modified (AEL-SM-0131 ) method. Standard: AMG-Standard, lot 7-1195, 195 AGU/ml.

375μL底物(50mM乙酸钠中加入1％麦芽糖，pH4.3)37℃温育5分钟。添加25μL的酶的乙酸钠稀释液。10分钟后，加入100μL0.25MNaOH终止反应。取20μL转到96孔微滴板上，加入200μL GOD-Perid溶液。室温30分钟后，测定650nm吸光度，参照AMG标准品以AGU/mg计算活性。375 μL of substrate (1% maltose in 50 mM sodium acetate, pH 4.3) was incubated at 37° C. for 5 minutes. Add 25 µL of the enzyme dilution in sodium acetate. After 10 minutes, the reaction was terminated by adding 100 μL of 0.25M NaOH. Transfer 20 μL to a 96-well microtiter plate and add 200 μL GOD-Perid solution. After 30 minutes at room temperature, the absorbance at 650 nm was measured, and the activity was calculated as AGU/mg with reference to the AMG standard.

活性(AGU/ml)除以蛋白浓度(mg/ml)可算得以AGU/mg表示的比活。The specific activity expressed in AGU/mg was calculated by dividing the activity (AGU/ml) by the protein concentration (mg/ml).

曲霉的转化(一般方法)Transformation of Aspergillus (general method)

以米曲霉孢子接种100ml YPD(Sherman等，(1981)，酵母遗传学方法，冷泉港实验室)，振荡培养约24小时。通过微孔布过滤收集菌丝体，用200ml0.6MMgSO4清洗。将菌丝体悬浮在15ml1.2M MgSO4，10mM NaH2PO4中，pH5.8中。将悬浮液于冰上冷却，加入1ml含120mg NovozymTM234的缓冲液。5分钟后，加入1ml 12mg/ml BSA(Sigma H25型)，37℃下轻微振荡连续培养1.5-2.5小时，直到通过镜检观察到样品中大量原生质体出现。100 ml of YPD (Sherman et al., (1981), Methods in Yeast Genetics, Cold Spring Harbor Laboratory) was inoculated with Aspergillus oryzae spores, and cultured with shaking for about 24 hours. Collect the mycelium by filtration through a microporous cloth and wash with 200 ml of 0.6 M MgSO 4 . The mycelium was suspended in 15 ml of 1.2M MgSO 4 , 10 mM NaH 2 PO 4 , pH 5.8. The suspension was cooled on ice and 1 ml of buffer containing 120 mg of Novozym ™ 234 was added. After 5 minutes, 1ml of 12mg/ml BSA (Sigma H25 type) was added, and cultured continuously at 37°C for 1.5-2.5 hours with slight shaking until a large number of protoplasts in the sample were observed by microscopy.

微孔布过滤悬浮液，滤液移至无菌试管，加5ml 0.6M山梨糖醇，100mMTris-HCl，pH7.0覆盖。1000g下离心5分钟，从MgSO4层上层收集原生质体。2倍体积的STC(1.2M山梨糖醇，10mM Tris-HCl，pH7.5，10mM CaCl2)加入到该原生质体悬浮液中，将该混合物1000g离心5分钟。将原生质体沉淀重悬在3ml STC中，重新沉积。重复该过程。最终，将原生质沉体沉淀重悬在0.2-1ml STC中。Filter the suspension with a microporous cloth, transfer the filtrate to a sterile test tube, add 5ml of 0.6M sorbitol, 100mM Tris-HCl, pH7.0 to cover. Centrifuge at 1000 g for 5 min to collect protoplasts from the upper layer of MgSO 4 layer. Two volumes of STC (1.2M sorbitol, 10mM Tris-HCl, pH 7.5, 10mM CaCl 2 ) was added to the protoplast suspension, and the mixture was centrifuged at 1000 g for 5 minutes. Resuspend the protoplast pellet in 3 ml STC and redeposit. Repeat the process. Finally, resuspend the protoplast pellet in 0.2-1 ml STC.

将100μL原生质体悬浮液与5-25μg p3SR2(Hynes等，Mol.And Cel.Biol.，Vol.3，No.8，1430-1439，Aug.1983所述携带构巢曲霉amdS基因的质粒)混合在10μLSTC中。混合液室温静置25分钟。加入0.2ml 60％PEG4000(BDH29576)，10 mM CaCl2，10mMTris-HCl，pH7.5，小心混合(两次)，最终加入0.85ml同一溶液，小心混合。使该混合液室温静置25分钟，2500g下旋转15分钟，得到的沉淀重悬于2ml 1.2M山梨糖醇中。再次沉淀后，将原生质体铺板在基本平板(Cove，(1996)，Biochem.Biophys.Acta113，51-56)上，该平板中含有1.0M蔗糖，pH7.0，10mM乙酰胺作氮源，20mM CsCl抑制背景生长。37℃培育4-7天后，拣出孢子，悬在无菌水中，划线接种以获得单菌落。重复该过程，第二次再分离后取单菌落保存为确定的转化体。100 μL of the protoplast suspension was mixed with 5-25 μg of p3SR2 (the plasmid carrying the A. nidulans amdS gene described by Hynes et al., Mol.And Cel.Biol., Vol.3, No.8, 1430-1439, Aug.1983) in 10 μL STC. The mixture was allowed to stand at room temperature for 25 minutes. 0.2ml 60% PEG4000 (BDH29576), 10mM CaCl2, 10mM Tris-HCl, pH 7.5 were added, mixed carefully (twice), and finally 0.85ml of the same solution was added, mixed carefully. The mixture was allowed to stand at room temperature for 25 minutes, spun at 2500 g for 15 minutes, and the resulting pellet was resuspended in 2 ml of 1.2M sorbitol. After re-precipitation, the protoplasts were plated on a basic plate (Cove, (1996), Biochem.Biophys.Acta113, 51-56), containing 1.0M sucrose, pH7.0, 10mM acetamide as nitrogen source, 20mM CsCl inhibits background growth. After incubation at 37°C for 4-7 days, the spores were picked out, suspended in sterile water, and streaked to obtain single colonies. This process was repeated, and after the second re-isolation, a single colony was taken and saved as a confirmed transformant.

补料分批发酵fed-batch fermentation

在含有以麦芽糖糊精为碳源，尿素为氮源，并含有酵母提取物的培养基中进行补料分批发酵。将所需米曲霉宿主细胞的摇瓶培养液接种到含3.5％所述碳源和0.5％所述氮源的培养基中进行补料分批发酵。在34℃pH5.0的条件下培养24小时后，开始连续补加碳源和氮源。保持碳源水平作为限制性因子，要确保氧过量供应。补料分批培养进行4天，然后通过离心，超滤，澄清过滤和除菌过滤来回收酶。Fed-batch fermentation was carried out in a medium containing maltodextrin as carbon source, urea as nitrogen source and yeast extract. Inoculate the shake flask culture fluid of the desired Aspergillus oryzae host cells into a medium containing 3.5% of the carbon source and 0.5% of the nitrogen source for fed-batch fermentation. After culturing for 24 hours under the condition of 34° C. and pH 5.0, the continuous supplementation of carbon source and nitrogen source was started. Keeping the carbon source level as the limiting factor ensures oxygen excess. Fed-batch culture was carried out for 4 days, and then the enzyme was recovered by centrifugation, ultrafiltration, clarification filtration and sterile filtration.

纯化purification

将培养液过滤，加入硫酸铵(AMS)到1.7M，pH调到5。离心除去沉淀的物质，将含有葡糖淀粉酶活性的溶液上样到Toyo Pearl Butyl柱，该柱已用1.7M AMS，20mM乙酸钠，pH5预平衡。用平衡缓冲液洗出未结合的物质。用10mM乙酸钠，pH4.5，以10倍柱体积中1.7-0M AMS的线性梯度洗脱结合蛋白。收集含葡糖淀粉酶的级分，并对20mM乙酸钠，pH4.5透析。然后将该溶液上样到Q Sepharose柱，该柱已用10mM Piperazin，Sigma，pH5.5预平衡。用平衡缓冲液洗下未结合的物质。以在10mM Piperazin中0-0.3M氯化钠(10倍柱体积)的线性梯度洗脱结合蛋白。收集含葡糖淀粉酶的级分，通过SDS-PAGE确认纯度。Filter the culture solution, add ammonium sulfate (AMS) to 1.7M, and adjust the pH to 5. The precipitated material was removed by centrifugation, and the solution containing glucoamylase activity was loaded onto a Toyo Pearl Butyl column, which had been pre-equilibrated with 1.7M AMS, 20mM sodium acetate, pH5. Unbound material was washed out with equilibration buffer. Bound proteins were eluted with a linear gradient of 1.7-0 M AMS in 10 column volumes with 10 mM sodium acetate, pH 4.5. Fractions containing glucoamylase were pooled and dialyzed against 20 mM sodium acetate, pH 4.5. This solution was then loaded onto a Q Sepharose column which had been pre-equilibrated with 10 mM Piperazin, Sigma, pH 5.5. Unbound material was washed with equilibration buffer. Bound protein was eluted with a linear gradient of 0-0.3M NaCl (10 column volumes) in 10 mM Piperazin. Fractions containing glucoamylase were collected and their purity was confirmed by SDS-PAGE.

T1/2(半衰期)方法IT 1/2 (half-life) Method I

变体的热稳定性使用下列方法测定为T1/2：68℃、70℃或75℃下将950μL50mM乙酸钠缓冲液(pH4.3)(NaOAc)温育5分钟。缓冲液中加入50μL酶(4AGU/ml)。在，例如0，5，10，20，30和40分钟时取样2×40μL样品，冰上冷却。用温育前(0分钟)测定的活性(AGU/ml)为参照(100％)。稳定性的降低(百分比)计算为保温时间的函数。在不同时间测定葡糖淀粉酶的百分比残余活性。T1/2表示相对活性降为50％所需的时间。The thermal stability of the variants was determined as T 1/2 using the following method: 950 μL of 50 mM sodium acetate buffer (pH 4.3) (NaOAc) was incubated at 68°C, 70°C or 75°C for 5 minutes. Add 50 μL of enzyme (4 AGU/ml) to the buffer. At, eg, 0, 5, 10, 20, 30 and 40 minutes, 2 x 40 [mu]L samples were taken and cooled on ice. The activity (AGU/ml) measured before incubation (0 min) was used as reference (100%). The decrease in stability (percentage) was calculated as a function of incubation time. The percent residual activity of the glucoamylase was determined at different times. T 1/2 represents the time required for the relative activity to drop to 50%.

T1/2(半衰期)方法IIT 1/2 (half-life) Method II

T1/2测定步骤如下：在30％葡萄糖，50mM乙酸钠，pH4.5，所需温度(例如70℃)下温育目标酶(约0.2 AGU/ml)。在设定的时间间隔取样，冰上冷却，用pNPG方法(如下所述)测定残余酶活性。The T 1/2 assay procedure is as follows: incubate the target enzyme (about 0.2 AGU/ml) in 30% glucose, 50 mM sodium acetate, pH 4.5, at a desired temperature (eg 70° C.). Samples were taken at set time intervals, cooled on ice, and residual enzyme activity was determined by the pNPG method (described below).

在不同的时间测定葡糖淀粉酶的百分比残余活性。T1/2表示相对活性降为50％所需的时间。The percent residual activity of the glucoamylase was determined at different times. T 1/2 represents the time required for the relative activity to drop to 50%.

残余酶活的测定(pNPG法)Determination of residual enzyme activity (pNPG method)

pNPG试剂：pNPG reagent:

0.2g pNPG(对-硝基苯葡萄糖吡喃糖苷)溶解在0.1M乙酸盐缓冲液(pH4.3)中，补足100ml。0.2 g of pNPG (p-nitrophenylglucopyranoside) was dissolved in 0.1 M acetate buffer (pH 4.3) to make up 100 ml.

硼酸盐溶液：Borate solution:

3.8g Na2B4O7·10H2O溶解在Milli-Q water中，补足100ml。Dissolve 3.8g Na 2 B 4 O 7 ·10H 2 O in Milli-Q water and make up 100ml.

25μL样品中加入50μL底物，50℃下温育2小时。加入150μL硼酸盐溶液终止反应。405nm测定光密度，计算残余活性。Add 50 μL substrate to 25 μL sample and incubate at 50°C for 2 hours. Add 150 μL of borate solution to stop the reaction. The optical density was measured at 405nm, and the residual activity was calculated.

pAMGY的构建Construction of pAMGY

pAMGY载体如下构建：用AMG基因取代pJSO026中的脂肪酶基因，所述AMG基因是用正向引物FG2：5’-CAT CCC CAG GAT CCT TAC TCAGCA ATG-3’(SEQ ID NO：10)和反向引物RG2：5’-CTC AAA CGA CTCACC AGC CTC TAG AGT-3’(SEQ ID NO：11)，以AMG基因的质粒pLAC103为模板经PCR扩增获得。用XbaI和SmaI在37℃消化pJSO026质粒2小时，用Klenow片段使PCR扩增子末端钝化，然后用XbaI消化。连接该载体片段和PCR扩增子，通过电转化作用转化进入大肠杆菌。产生的载体为pAMGY。The pAMGY vector was constructed as follows: the lipase gene in pJSO026 was replaced with the AMG gene, which was made with the forward primer FG2: 5'-CAT CCC CAG GAT CCT TAC TCAGCA ATG-3' (SEQ ID NO: 10) and the reverse The primer RG2: 5'-CTC AAA CGA CTCACC AGC CTC TAG AGT-3' (SEQ ID NO: 11) was obtained by PCR amplification using the plasmid pLAC103 of the AMG gene as a template. The pJSO026 plasmid was digested with XbaI and SmaI at 37°C for 2 hours, the ends of the PCR amplicon were blunted with Klenow fragment, and then digested with XbaI. The vector fragment and the PCR amplicon were ligated and transformed into Escherichia coli by electroporation. The resulting vector was pAMGY.

pLaC103的构建Construction of pLaC103

用黑曲霉AMGII cDNA克隆(Boel等，(1984)，同上)作为构建pLaC103的来源以便在酿酒酵母中表达AMG的GII形式。The A. niger AMGII cDNA clone (Boel et al., (1984), supra) was used as a source for the construction of pLaC103 for expression of the GII form of AMG in S. cerevisiae.

构建分几步，列于下：The construction is divided into several steps, listed below:

用XbaI切割pT7-212(EP3 7856/US5162498)，用Klenow DNA聚合酶和dNTP进行末端钝化。用EcoRI切割后，得到的载体片段通过琼脂糖凝胶电泳纯化，与pBoe153的2.05kb EcoR1-EcoRV片段连接，从而在所得质粒pG2x的AMG编码片段的EcoRV末端再造XbaI位点。pT7-212 (EP3 7856/US5162498) was cut with XbaI and end blunted with Klenow DNA polymerase and dNTPs. After cleavage with EcoRI, the resulting vector fragment was purified by agarose gel electrophoresis and ligated with the 2.05 kb EcoR1-EcoRV fragment of pBoel53 to recreate an XbaI site at the EcoRV terminus of the AMG-encoding fragment of the resulting plasmid pG2x.

为除去AMG cds上游的DNA，以及用适当的限制性内切酶识别位点装饰AMG的编码DNA，进行下述的构建：To remove the DNA upstream of the AMG cds, and to decorate the AMG-encoding DNA with appropriate restriction enzyme recognition sites, the following constructions were performed:

分离p53的930kb EcoRI-PstI片段并进行AluI切割，将得到的771bpAlu-PstI片段与带有末端钝化型EcoRI位点的pBR322(如上)连接，再用PstI切割。在得到的质粒pBR-AMG’中，在距离AMG cds的起始密码子34bp处再造EcoRI位点。The 930kb EcoRI-PstI fragment of p53 was isolated and cut with AluI, and the resulting 771bpAlu-PstI fragment was ligated with pBR322 (as above) with a blunt-ended EcoRI site and cut with PstI. In the resulting plasmid pBR-AMG', an EcoRI site was recreated at a distance of 34 bp from the start codon of the AMG cds.

从pBR-AMG’分离得到775bp的EcoRI-PstI片段，并在包括pT7-212的XbaI-EcoRI载体片段的连接反应中与pG2x中的1151bp PstI-XbaI片段连接。A 775 bp EcoRI-PstI fragment was isolated from pBR-AMG' and ligated to the 1151 bp PstI-XbaI fragment in pG2x in a ligation reaction involving the XbaI-EcoRI vector fragment of pT7-212.

得到的质粒pT7GII在有碱性磷酸酶的存在下进行BamHI切割，然后在磷酸酶失活后用SphI进行部分切割。得到2489bp的SphI-BamHI片段，其包括连接到AMGII cds的S.c.TPI启动子。上述片段与pT7GII的1052bpBamHI片段一起与经SphI-BamHI消化及碱性磷酸酶处理的pMT743(EP37856/US5162498)载体片段连接。得到的质粒是pLaC103。The resulting plasmid pT7GII was cut with BamHI in the presence of alkaline phosphatase and then partially cut with SphI after inactivation of the phosphatase. A SphI-BamHI fragment of 2489 bp was obtained, which included the S.c.TPI promoter linked to the AMGII cds. The above fragment and the 1052 bp BamHI fragment of pT7GII were ligated with the pMT743 (EP37856/US5162498) vector fragment digested with SphI-BamHI and treated with alkaline phosphatase. The resulting plasmid was pLaC103.

筛选热稳定的AMG变体Screening for thermostable AMG variants

在下述热稳定性滤膜分析中筛选文库。Libraries were screened in the thermostability filter assay described below.

用于热稳定性的滤膜分析Membrane Analysis for Thermal Stability

酵母文库铺板在含100μg/ml氨苄青霉素的SCFura琼脂板上的醋酸纤维素滤膜(OE67，Schleicher&Schuell，Dassel，德国)-和硝酸纤维素滤膜(Protran-Ba 85，Schleicher&Schuell，Dassel，德国)的夹层上，30℃下至少72小时。将菌落影印到已用甲醇活化1分钟的PVDF滤膜(Immobilon-P，Millipore，Bedford)，或Protran滤膜(未活化)上，然后用0.1M NaAc洗膜，再室温下温育2小时。用自来水从PVDF/Protran滤膜上洗下菌落。温育前用针分别标记每一滤膜夹层和PVDF/Protran滤膜，这样在筛选后可以在滤膜上定位阳性变体。将结合有变体的PVDF膜转移到有0.1M NaAc，pH4.5的容器中，47℃或使用Protran膜时可选67-69℃下温育15分钟。将SCura-琼脂平板上的醋酸纤维素和硝酸纤维素滤膜夹层室温保存以备用。温育后，在含5％麦芽糖，1％琼脂糖，50mMNaAc，pH4.5的平板上测定残余活性。含有PVDF的试验板如滤膜夹层一样标记，并50℃温育2小时。除去PVDF滤膜后，试验板用Glucose GOD perid(Boehringer Mannheim GmbH，德国)染色。具有残余活性的变体在试验板上表现为白色背景上的墨绿点。改进的变体位于保存板上。按照第一次筛选的条件再次筛选改进的变体。Yeast libraries were plated on cellulose acetate filters (OE67, Schleicher & Schuell, Dassel, Germany)- and nitrocellulose filters (Protran-Ba 85, Schleicher & Schuell, Dassel, Germany) on SCFura agar plates containing 100 μg/ml ampicillin. On the interlayer, at 30°C for at least 72 hours. Colonies were replicated onto PVDF filters (Immobilon-P, Millipore, Bedford) activated with methanol for 1 minute, or Protran filters (unactivated), washed with 0.1M NaAc, and incubated at room temperature for 2 hours. Colonies were washed from the PVDF/Protran filters with tap water. Each filter sandwich and PVDF/Protran filter were individually labeled with a needle prior to incubation so that positive variants could be localized on the filter after selection. Transfer the variant-bound PVDF membrane to a container with 0.1M NaAc, pH 4.5, and incubate for 15 minutes at 47°C or optionally 67-69°C when using a Protran membrane. The cellulose acetate and nitrocellulose filter sandwiches on SCura-agar plates were stored at room temperature for later use. After incubation, residual activity was determined on plates containing 5% maltose, 1% agarose, 50 mM NaAc, pH 4.5. Assay plates containing PVDF were labeled as filter sandwiches and incubated at 50°C for 2 hours. After removal of the PVDF filter, the test plate was stained with Glucose GOD perid (Boehringer Mannheim GmbH, Germany). Variants with residual activity appear on the assay plate as dark green spots on a white background. Improved variants are on save boards. Improved variants were screened again against the criteria of the first screen.

使用DOPE程序进行随机诱变的一般方法General method for random mutagenesis using the DOPE procedure

随机诱变可按下列步骤进行：Random mutagenesis can be performed as follows:

1.在亲本酶中选择用于修饰的目的区域，1. Select the target region for modification in the parent enzyme,

2.在所选目的区域中确定突变和非突变位点，2. Determine the mutation and non-mutation sites in the selected target region,

3.确定可以进行何种突变，例如考虑所要构建的变体的所需稳定性和/或性能，3. Determining what mutations can be made, e.g. considering the required stability and/or properties of the variant to be constructed,

4.挑选结构合理的突变，4. Select structurally sound mutations,

5.参照步骤4调整经步骤3挑选的残基，5. Refer to step 4 to adjust the residues selected in step 3,

6.通过使用适当的DOPE算法分析核苷酸分布，6. Analyze the nucleotide distribution by using the appropriate DOPE algorithm,

7.如果需要，调整所要残基以满足遗传密码的实用性，例如考虑由遗传密码引起的约束，如为避免引入终止密码子所引起的约束；本领域技术人员可以知道一些密码子组合并不实用，需要进行调整。7. If necessary, adjust the desired residues to meet the practicality of the genetic code, such as taking into account the constraints caused by the genetic code, such as to avoid the constraints caused by the introduction of stop codons; those skilled in the art will know that some codon combinations do not Practical, needs tweaking.

8.制备引物8. Preparation of Primers

9.使用引物进行随机诱变9. Random Mutagenesis Using Primers

10.通过筛选所需改进的特性来选择所得葡糖淀粉酶变体。10. Select the resulting glucoamylase variants by screening for the desired improved properties.

Dope算法Dope algorithm

适用于步骤6的Dope算法为本领域所熟知。Tomandl，D.等，1997，计算机辅助分子设计杂志(Joumal of Computer-Aided Molecular Design)11：29-38记述一种这样的算法。另一算法是DOPE(Jensen，LJ，Andersen，KV，Sevendsen，A，和Kretzschmar，T(1998)核酸研究26：697-702)。The Dope algorithm suitable for use in step 6 is well known in the art. Tomandl, D. et al., 1997, Journal of Computer-Aided Molecular Design 11:29-38 describe one such algorithm. Another algorithm is DOPE (Jensen, LJ, Andersen, KV, Sevendsen, A, and Kretzschmar, T (1998) Nucleic Acids Res. 26:697-702).

                         实施例Example

实施例1Example 1

AMGG2变体的构建Construction of AMGG2 variants

定点诱变site-directed mutagenesis

为构建AMG G2酶(SEQ ID NO：2)的变体，参照制造商说明书使用市售试剂盒，Chameleon双链定点诱变试剂盒。To construct variants of the AMG G2 enzyme (SEQ ID NO: 2), a commercially available kit, the Chameleon double-strand site-directed mutagenesis kit, was used according to the manufacturer's instructions.

将编码目的AMG G2酶的基因定位在pMT838上，所述质粒是在含AMG G1型的质粒pCAMG91(见图1)上缺失G2 nt.1362和G2 nt.1530间的DNA而得到的。The gene encoding the AMG G2 enzyme of the purpose is located on pMT838, and the plasmid is obtained by deleting the DNA between G2 nt.1362 and G2 nt.1530 on the plasmid pCAMG91 (seeing Figure 1) containing the AMG G1 type.

参照制造商说明书，利用下列引物将pMT838的氨苄青霉素基因的ScaI位点改变为Mlul位点：Following the manufacturer's instructions, the ScaI site of the ampicillin gene of pMT838 was changed to a Mlul site using the following primers:

7258：5’p gaa tga ctt ggt tga cgc gtc acc agt cac 3’(SEQ ID NO：3)7258: 5'p gaa tga ctt ggt tga cgc gtc acc agt cac 3' (SEQ ID NO: 3)

(这样就改变了氨苄青霉素抗性基因中发现并用于切割至MluI位点的ScaI位点)。然后以含目的AMG基因的pMT838载体作DNA聚合酶的模板并使用oligo 7258(SEQ ID NO：3)及21401(SEQ ID NO：4)。(This alters the ScaI site found in the ampicillin resistance gene and used for cleavage to the MluI site). Then use the pMT838 vector containing the target AMG gene as a template for DNA polymerase and use oligo 7258 (SEQ ID NO: 3) and 21401 (SEQ ID NO: 4).

引物21401(SEQ ID NO：4)用作选择性引物。Primer 21401 (SEQ ID NO: 4) was used as a selective primer.

21401：5’pgg gga tca tga tag gac tag cca tat taa tga agg gca tat acc acg ccttgg acc tgc gtt ata gcc 3’21401: 5’pgg gga tca tga tag gac tag cca tat taa tga agg gca tat acc acg ccttgg acc tgc gtt ata gcc 3’

(在未改变氨基酸序列的情况下，改变了AMG基因中发现的ScaI位点)。(The ScaI site found in the AMG gene was altered without altering the amino acid sequence).

通过加入含有所要突变的适合的oligo可引入所要的突变(例如，引入半胱氨酸残基)到目的AMG基因中。Desired mutations (eg, introducing cysteine residues) can be introduced into the AMG gene of interest by adding an appropriate oligo containing the desired mutation.

用引物107581引入T12PIntroduction of T12P with primer 107581

107581：5’pgc aac gaa gcg ccc gtg gct cgt ac 3’(SEQ ID NO：5)107581: 5' pgc aac gaa gcg ccc gtg gct cgt ac 3' (SEQ ID NO: 5)

通过整个基因的测序证实突变。使用上述“材料和方法”部分的方法将质粒转化进入米曲霉。按照上述变体“材料和方法”部分所述进行变体的发酵及纯化。Mutations were confirmed by sequencing of the entire gene. Plasmids were transformed into A. oryzae using the method described in the "Materials and Methods" section above. Fermentation and purification of the variants were performed as described above in the "Materials and Methods" section for the variants.

实施例2Example 2

通过定域随机掺入诱变来构建与亲本酶相比稳定性提高了的黑曲霉AMG变体Construction of Aspergillus niger AMG variants with improved stability compared to the parental enzyme by localized random incorporation mutagenesis

为改进黑曲霉AMG的热稳定性，在预选区域进行随机突变。To improve the thermostability of Aspergillus niger AMG, random mutations were performed in preselected regions.

残基Residues

区域：    L19-G35Area: L19-G35

区域：    A353-V374Area: A353-V374

用DOPE软件(见材料与方法)在上述区域确定每一所需改变所要掺入的密码子以使终止密码子的数量最少(参见表1)。在密码子的三个位置计算确切的核苷酸分布以给出建议的氨基酸变化群。所要掺入的区域特别掺入在指定的位置，这可以有较多的机会获得所要残基，但是也有其它可能。The codons to be incorporated for each desired change were determined in the above regions using DOPE software (see Materials and Methods) to minimize the number of stop codons (see Table 1). The exact nucleotide distribution is calculated at the three positions of the codon to give a suggested population of amino acid changes. Regions to be incorporated are specifically incorporated at designated positions, which may have a higher chance of obtaining the desired residue, but other possibilities are also possible.

第一列是要突变的氨基酸，第二列是野生型的百分比，第三列是新的氨基酸。The first column is the amino acid to be mutated, the second column is the percentage of wild type, and the third column is the new amino acid.

表1：Table 1:

在L19-G35中的掺入Incorporation in L19-G35

L19 90％  NL19 90% N

N20 95％  TN20 95% T

N21不变N21 unchanged

I22 不变I22 unchanged

G23 95％  AG23 95% A

A24 90％  S，TA24 90% S, T

D25 93％  S，T，RD25 93% S, T, R

G26 95％  AG26 95% A

A27 90％  S，TA27 90% S, T

W28＜80％R，YW28<80% R, Y

V29不变V29 unchanged

S30 93％  T，NS30 93% T, N

G31 95％  AG31 95% A

A32 95％  VA32 95% V

D33 80％  R，K，HD33 80% R, K, H

S34 90％  NS34 90% N

G35不变G35 unchanged

得到的掺入寡核苷酸链在表2中作为有义链表示：表中还有引物序列，野生型核苷酸序列，亲本氨基酸序列和每一掺入位置的核苷酸分布。The resulting incorporated oligonucleotide strands are represented as the sense strand in Table 2: Also included in the table are the primer sequences, wild-type nucleotide sequence, parental amino acid sequence and nucleotide distribution at each incorporated position.

表2：位点：          19     20   21     22     23   24   25      26     27氨基酸序列：    L      N    N      I      G    A    D       G      A引物：          12T    A3T  AAC    ATC    G4G  5CG  67C     G4T    8CT野生型序列：    CTG    AAT  AAC    ATC    GGG  GCG  GAC     GGT    GCT位点(续)：      28     29   30     31       32     33         34      35氨基酸序列(续)：W      V    S      G        A      D          S       G引物：          91010  GTG  1112C  G4C      G13G   141516     1718T   GGC野生型序列：    TGG    GTG  TCG    GGC      GCG    GAC        TCT     GGC每一掺入位置的核苷酸分布1：A10，C902：A6，T943：A95，C54：G95，C55：G91，A3，T3，C36：G95，A3，C27：G3，A95，C28：G92，A4，T49：A3，T9710：G95，T511：G3，A9712：G95，A2，C313：T5，C9514：G88，A8，C415：G7，A9316：G4，C9617：G4，A9618：G95，A2，C3正向引物(SEQ ID NO：6)：FAMGII′ 5-C GAA GCG ACC GTG GCT CGT ACT GCC ATC 12T A3T AAC ATCG4G 5CG  67C G4T 8CT  91010 GTG 1112C G4C G13G  141516 1718T GGCATT GTC GTT GCT AGT CCC AGC ACG GAT AAC-3′反向引物(SEQ ID NO：7)：Table 2: Siter: 19 20 21 22 23 24 25 26 27 Amino acid sequence: l n i g a d g a primer: 12t a3T AAC ATC G4G 5CG 67C G4T 8CT Wild Strike GCT site (sequel): 28 29 30 32 32 33 34 35 amino acid sequence (sequel): W v s g A D S g primer: 91010 GTG 1112C G4C G4C G13G 141516 1718T GGC wild -type sequence: TGG GTG TCG GCG GCG GCG GCG GCG GAC TCT Nucleotide distribution at each incorporation position of GGC 1: A10, C902: A6, T943: A95, C54: G95, C55: G91, A3, T3, C36: G95, A3, C27: G3, A95, C28: G92 , A4, T49: A3, T9710: G95, T511: G3, A9712: G95, A2, C313: T5, C9514: G88, A8, C415: G7, A9316: G4, C9617: G4, A9618: G95, A2, C3 Positive primer (SEQ ID NO: 6): FAMGII ′ 5-C GAA GCG ACC GCT CGT CGT GCC ATC 12T ACGG4G 5CG 47C G4T 8CT 91010C G4C G4C G13G 141518T GGCACAC GTC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC -3' reverse primer (SEQ ID NO: 7):

RAMG1：5′-GAT GGC AGT ACG AGC CAC GGT CGC TTC G-3′RAMG1: 5′-GAT GGC AGT ACG AGC CAC GGT CGC TTC G-3′

表3table 3

在区域A353-V374中的掺入：Incorporation in the region A353-V374:

A353    ＜80％     D，E，Q，N，YA353 ＜80％ D, E, Q, N, Y

L354    90％      Q，EL354 90% Q, E

Y355    90％      N，QY355 90% N, Q

S356    90％      T，D，NS356 90% T, D, N

G357    80％      P，A，S，TG357 80% P, A, S, T

A358    93％      SA358 93% S

A359    90％      S，T，NA359 90% S, T, N

T360    90％      R，KT360 90% R, K

G361    85％      A，S，TG361 85% A, S, T

T362    90％      ST362 90% S

Y363    不变Y363 unchanged

S364    93％      DS364 93% D

S365    93％      N，Q，KS365 93% N, Q, K

S366    93％      P，DS366 93% P, D

S367    不变S367 unchanged

S368    93％      D，N，TS368 93% D, N, T

Y369    93％      Q，EY369 93% Q, E

Y370    不变Y370 unchanged

S371    93％      NS371 93% N

S372    93％      N，TS372 93% N,T

I373    不变I373 unchanged

V374    93％      N，Y，HV374 93% N, Y, H

得到的掺入寡核苷酸链在表4中作为有义链表示：表中还有引物序列，野生型核苷酸序列，亲本氨基酸序列和每一掺入位置的核苷酸分布。The resulting incorporated oligonucleotide strands are represented as the sense strand in Table 4: Also included in the table are the primer sequences, wild-type nucleotide sequence, parental amino acid sequence and nucleotide distribution at each incorporated position.

表4：位点：      353 354 355 356 357  358  359       360    361   362氨基酸序列：A   L   Y   S   D    A    A         T      G     T引物：      123 45A 6AC 78C 910T 11CT  1213T    1415A  1617C 18CC野生型序列：GCA CTG TAC AGC GAT   GCT   GCT      ACT   GGC   ACC位点(续)：          363 364   365    366   367   368    369Table 4: Siter: 353 354 355 357 357 358 360 360 361 362 Amino acid Serial: A L Y Y S D A T g T Primary: 123 45A 6AC 78C 910T 11CT 1213T 1417C 18CC Wildlord Sequence: GCA CTG TAC AGC GAT GCT GCT ACT GGC ACC loci (continued): 363 364 365 366 367 368 369

370氨基酸序列(续)：    Y   S     S      S     S     S      T      Y引物(续)：          TAC 1920T A2122 2324C AGT   1425C 2627G T28T野生型序列(续)：    TAC TCT   TCG    TCC  AGT    TCG   ACT  TAT位点(续)：          371   372   373 374氨基酸序列(续)：    S     S     I   V引物(续)：          A16T  2930T ATT 313233野生型序列(续)：    AGT   AGC   ATT GTA每一掺入位置的核苷酸分布1：G91，A3，T3，C32：A13，C873：A40，T604：G3，A3，C945：A6，T946：G4，A4，T927：G2，A96，C28：G93，A3.5，C3.59：G87，A8，C510：A84，C1611：G93，T712：G92，A5，T313：A3，C9714：G3，A9715：G2，A2，T4，C9216：G93，A717：G93，C718：A90，T1019：G4，A9620：G95，A521：G96，A422：G3，C9723：G2，A1，T95，C224：A3，C97370 amino acid sequence (sequel): Y s s s s s s t y primer (continued): TAC 1920t A2122 2324C AGT 1425C 2627G T28T wild -type sequence (sequel): TAC TCT TCG TCC AGT TCG ACT TAT site (continued): 371 372 373 374 amino acid sequence (continued): S S S I V Primer (continued): A16T 2930T ATT 313233 wild-type sequence (continued): AGT AGC ATT GTA Nucleotide distribution at each incorporation position 1: G91, A3, T3 , C32: A13, C873: A40, T604: G3, A3, C945: A6, T946: G4, A4, T927: G2, A96, C28: G93, A3.5, C3.59: G87, A8, C510: A84 , C1611: G93, T712: G92, A5, T313: A3, C9714: G3, A9715: G2, A2, T4, C9216: G93, A717: G93, C718: A90, T1019: G4, A9620: G95, A521: G96 , A422: G3, C9723: G2, A1, T95, C224: A3, C97

25：G95，A3，C225: G95, A3, C2

26：G2，A96，C226: G2, A96, C2

27：A5，C9527: A5, C95

28：A95，T528: A95, T5

29：G2，A9829: G2, A98

30：G94，A4，C230: G94, A4, C2

31：G94，A3，T1，C231: G94, A3, T1, C2

32：A4，T9632: A4, T96

33：A20，C80引物：FAMGIV(SEQ ID No：8)5′-GTG TCG CTG GAC TTC TTC AAG 123 45A 6AC 78C 910T 11CT 1213T1415A 1617C 18CC TAC 1920T A2122 2324C AGT 1425C 2627G T28TA16T 2930C ATT 313233 GAT GCC GTG AAG ACT TTC GCC GA-3′33：A20，C80引物：FAMGIV(SEQ ID No：8)5′-GTG TCG CTG GAC TTC TTC AAG 123 45A 6AC 78C 910T 11CT 1213T1415A 1617C 18CC TAC 1920T A2122 2324C AGT 1425C 2627G T28TA16T 2930C ATT 313233 GAT GCC GTG AAG ACT TTC GCC GA-3′

引物RAMGVI(SEQ ID NO：9)5′ctt gaa gaa gtc cag cga cac-3′Primer RAMGVI (SEQ ID NO: 9) 5'ctt gaa gaa gtc cag cga cac-3'

随机诱变random mutagenesis

使用表2和表3中可见的掺入的寡核苷酸(通称FAMG)和针对L19-G35区域的反向引物RAMG，以及，覆盖了N-末端(FG2：5’-CAT CCC CAGGAT CCT TAC TCA GCA ATG-3’(SEQ ID NO：10)和C-末端(RG2：5’-CTCAAA CGA CTC ACC AGC CTC TAG AGT(SEQ ID NO：11)的特定SEQ IDNO：2引物，通过重叠延伸方法(Horton等，基因，77(1989)，pp.61-68)产生PCR-文库片段，有21个碱基对的重叠。质粒pAMGY用作聚合酶链式反应的模板。在大肠杆菌/酵母穿梭载体pAMGY中通过同源重组克隆PCR片段(参见材料和方法)。Using the incorporated oligonucleotides (commonly known as FAMG) seen in Tables 2 and 3 and the reverse primer RAMG for the L19-G35 region, and, covering the N-terminus (FG2: 5'-CAT CCC CAGGAT CCT TAC Specific SEQ ID NO: 2 primers for TCA GCA ATG-3' (SEQ ID NO: 10) and C-terminal (RG2: 5'-CTCAAA CGA CTC ACC AGC CTC TAG AGT (SEQ ID NO: 11) by overlap extension method (Horton et al., Gene, 77 (1989), pp.61-68) generated PCR-library fragments with an overlap of 21 base pairs. Plasmid pAMGY was used as a template for the polymerase chain reaction. In E. coli/yeast shuttle The PCR fragment was cloned by homologous recombination in the vector pAMGY (see Materials and Methods).

筛选filter

如上述“材料和方法”部分所述，使用Protran膜在热稳定膜分析中筛选文库，并在67-69℃下温育。Libraries were screened in a thermostable membrane assay using Protran membranes and incubated at 67-69°C as described in the "Materials and methods" section above.

实施例2Example 2

在68℃的热稳定性Thermal Stability at 68°C

使用实施例1所述方法构建AMG G2变体。The AMG G2 variant was constructed using the method described in Example 1.

使用“材料和方法”中的方法I在68℃下测定热稳定性，以T1/2表示，并与相同条件下的野生型黑曲霉AMG G2对比。     酶   T   AMG G2野尘型   8.5   T72I+A246T   11.3   A495P   11.0   A425T+S465P+E408R+A495T   8.6   T379A+S386K+A393R+T2E   18.4   L66V+S394P+Y402F+T2R+RL   11.1   S386R+A393R+T2R   14.1   S386N+E408R   12.6   A1V+L66R+Y402F+N427S+S486G T2K+S30P+N427M+S444G+V470MA393R+T490A+V59A+PLASD(N-末端延伸)S119P+Y312Q+Y402F+S416H，T379A+S386K+A393R+T2E，S386P+S340G+D357S+T360V.Thermostability was determined at 68°C using method I in Materials and Methods, expressed as T 1/2 , and compared to wild-type A. niger AMG G2 under the same conditions. enzyme T AMG G2 Wild Dust 8.5 T72I+A246T 11.3 A495P 11.0 A425T+S465P+E408R+A495T 8.6 T379A+S386K+A393R+T2E 18.4 L66V+S394P+Y402F+T2R+RL 11.1 S386R+A393R+T2R 14.1 S386N+E408R 12.6 A1V+L66R+Y402F+N427S+S486G T2K+S30P+N427M+S444G+V470MA393R+T490A+V59A+PLASD (N-terminal extension) S119P+Y312Q+Y402F+S416H, T379A+S386K+A393R+T2E, S386P+S340G+D357S+T360V.

实施例3Example 3

比活Bi live

AMG G2变体如实施例1构建。如“材料和方法”部分所述，在37℃pH4.5下测定比活为kcat或AGU/mg，使用麦芽糖为底物。     酶   AGU/mg  KCat(Sec.-1) AMG G2(野生型)I189T+Y223F+F227Y+Y402F+S119P     5.69.3 AMG G2 variants were constructed as in Example 1. Specific activities were determined as k cat or AGU/mg at 37°C pH 4.5 using maltose as substrate as described in the "Materials and methods" section. enzyme AGU/mg KCat(Sec.-1) AMG G2 (wild type) I189T+Y223F+F227Y+Y402F+S119P 5.69.3

实施例4Example 4

在75℃的热稳定性Thermal Stability at 75°C

采用实施例1所述方法构建AMG G2变体。The method described in Example 1 was used to construct the AMG G2 variant.

使用“材料和方法”中的方法I在75℃和pH4.5下测定热稳定性，以T1/2表示，并与相同条件下的野生型黑曲霉AMG G2对比。   AGRNo.   突变    Tx(分钟)   G2(对比)     4   136   V59A+A393R+T490A     6   109   S56A+V59A+N313G+S356G+A393R+S394R+Y402F     9   111   A11E+V59A+T72I+S119P+F237H+S240G+A246T+N313G+S340G+K352R+A393R+S394R-Y402F+E408R     10   120   T2H+A11P+V59A+T72I+S119P+A246T+N313G+D336S+T360V+A393R+Y402F+E408R+N427M     12   122   T2H+V59A+T72I+S119P+S240G+N313G+T360V+5368P+A393R+Y402F+E408R+N427M     10   124   N9A+S56A+V59A+S119P+A246T+N313G+E342T+A393R+S394R+Y402F+E408R     21   130   V59A+L66R+T72I+S119P+N313G+S340G+S3S6G+A393R+Y402F+E408R+N427M     29   132   T2H+N9A+V59A+S56A+L66R+T72I+S119P+N313G+F318Y+E342T+S356G+T390R+Y402F+E408R+N427M     9   141   T2H+A11E+V59A+S119P+N313G+E342T+S356P+A393R+S394I+Y402F+L410R+N427S     13   142   T2H+A11P+V59A+S119P+N313G+S340G+S356G+E408R+N427M     9   151   T2H+A11E+V59A+L66R+S119P+N313G+S340G+D3S7S+A393R+S394R+Y402F+E408R     20   154   T2H+N9A+S56A+V59A+L66R+T72I+S119P+S240G+N313G+S340G+K352R+A393R+S394R+Y402F+E408R+N427S     19 Thermostability was determined using method I in Materials and methods at 75 °C and pH 4.5, expressed as T 1/2 , and compared to wild-type A. niger AMG G2 under the same conditions. AGR No. mutation Tx(min) G2 (comparison) 4 136 V59A+A393R+T490A 6 109 S56A+V59A+N313G+S356G+A393R+S394R+Y402F 9 111 A11E+V59A+T72I+S119P+F237H+S240G+A246T+N313G+S340G+K352R+A393R+S394R-Y402F+E408R 10 120 T2H+A11P+V59A+T72I+S119P+A246T+N313G+D336S+T360V+A393R+Y402F+E408R+N427M 12 122 T2H+V59A+T72I+S119P+S240G+N313G+T360V+5368P+A393R+Y402F+E408R+N427M 10 124 N9A+S56A+V59A+S119P+A246T+N313G+E342T+A393R+S394R+Y402F+E408R twenty one 130 V59A+L66R+T72I+S119P+N313G+S340G+S3S6G+A393R+Y402F+E408R+N427M 29 132 T2H+N9A+V59A+S56A+L66R+T72I+S119P+N313G+F318Y+E342T+S356G+T390R+Y402F+E408R+N427M 9 141 T2H+A11E+V59A+S119P+N313G+E342T+S356P+A393R+S394I+Y402F+L410R+N427S 13 142 T2H+A11P+V59A+S119P+N313G+S340G+S356G+E408R+N427M 9 151 T2H+A11E+V59A+L66R+S119P+N313G+S340G+D3S7S+A393R+S394R+Y402F+E408R 20 154 T2H+N9A+S56A+V59A+L66R+T72I+S119P+S240G+N313G+S340G+K352R+A393R+S394R+Y402F+E408R+N427S 19

实施例5Example 5

AMG变体AGR130的糖化性能Glycation performance of AMG variant AGR130

如下所述在70℃测定具有改进的热稳定性的变体AGR130(V59A+L66R+T72I+S119P+N313G+S340G+S356G+A393R+Y402F+E408R+N427M)的糖化性能。The saccharification performance of the variant AGR130 with improved thermostability (V59A+L66R+T72I+S119P+N313G+S340G+S356G+A393R+Y402F+E408R+N427M) was determined at 70°C as follows.

参比酶是野生型黑曲霉AMG G2。糖化在下列条件下进行：The reference enzyme is wild type Aspergillus niger AMG G2. Saccharification is carried out under the following conditions:

底物    10DE麦芽糖糊精，约30％DS(w/w)Substrate 10DE maltodextrin, about 30% DS (w/w)

温度    70℃Temperature 70°C

初始pH  4.3(在70℃)Initial pH 4.3 (at 70°C)

酶量    0.24AGU/g DSEnzyme amount 0.24AGU/g DS

糖化saccharification

将麦芽糖糊精(从普通淀粉制备)溶解在煮沸的Milli-Q水中，制备糖化底物，将干物质调为约30％(w/w)。pH调到4.3。相当于15g干重的底物样品转移到50ml蓝盖玻璃烧瓶中，水浴搅拌。加入酶，必要时调pH。进行平行实验。定时取样，HPLC分析确定碳水化合物组成。A saccharification substrate was prepared by dissolving maltodextrin (prepared from common starch) in boiled Milli-Q water to bring the dry matter to about 30% (w/w). The pH was adjusted to 4.3. A substrate sample equivalent to 15 g dry weight was transferred to a 50 ml blue cap glass flask and stirred in a water bath. Add enzyme and adjust pH if necessary. Perform parallel experiments. Samples were taken regularly and analyzed by HPLC to determine the carbohydrate composition.

                    序列表<110>诺沃挪第克公司(Novo Nordisk A/S)<120>葡糖淀粉酶变体<130>5967.204-WO<160>13<170>适用于Windows 3.0的FastSEQ<210>1<211>1605<212>DNA<213>黑曲霉(Aspergillus niger)<220><221>sig-peptide<222>(1)...(72)<221>mat-peptide<222>(73)...(1602)<221>CDS<222>(1)...(1602)<400>1atg tcg ttc cga tct cta ctc gcc ctg agc ggc ctc gtc tgc aca ggg       48Met Ser Phe Arg Ser Leu Leu Ala Leu Ser Gly Leu Val Cys Thr GlySequence Listing <110> Novo Nordisk A/S <120> Glucoamylase Variant <130> 5967.204-WO <160>13 <170> FastSEQ for Windows 3.0 <210>1 <211>1605<212>DNA<213>Aspergillus niger<220><221>sig-peptide<222>(1)...(72)<221>mat-peptide<222>(73) ...(1602)<221>CDS<222>(1)...(1602)<400>1atg tcg ttc cga tct cta ctc gcc ctg agc ggc ctc gtc tgc aca ggg 48Met Ser Phe Arg Ser Leu Leu Ala Leu Ser Gly Leu Val Cys Thr Gly

            -20                 -15                 -10ttg gca aat gtg att tcc aag cgc gcg acc ttg gat tca tgg ttg agc       96Leu Ala Asn Val Ile Ser Lys Arg Ala Thr Leu Asp Ser Trp Leu Ser-20 -15 -10ttg GA AAT GTG Att TCC AAG CGC GCG ACC TTG GAT TGG TGG TGC 96leu Ala Asn Val Ile Serite

        -5                  1               5aac gaa gcg acc gtg gct cgt act gcc atc ctg aat aac atc ggg gcg      144Asn Glu Ala Thr Val Ala Arg Thr Ala Ile Leu Asn Asn Ile Gly Ala-5 1 5AAC GAA GCG Act CGT ACT GCC ATC CTG AAC ATC GGG GCG 144ASN GCG 144ASN GCG 144ASN GCG 144ASN GCG 144ASN GCG

10                  15                  20gac ggt gct tgg gtg tcg ggc gcg gac tct ggc att gtc gtt gct agt      192Asp Gly Ala Trp Val Ser Gly Ala Asp Ser Gly Ile Val Val Ala Ser25                 30                  35                   40ccc agc acg gat aac ccg gac tac ttc tac acc tgg act cgc gac tct       240Pro Ser Thr Asp Asn Pro Asp Tyr Phe Tyr Thr Trp Thr Arg Asp Ser10 15 20GAC GGT GCT GCT TCG GGC GCG GCG GCG GCG GAC TCT GGC ATT GTC GTT GTT GTT GTT GTT GTT GTT GTT 192asp Gly Ala Tr Val Ser Gly Ala Ala Val Val Val Ala AgCC AG GAC ACC TACC TATC TA that thesle gac tct 240Pro Ser Thr Asp Asn Pro Asp Tyr Phe Tyr Thr Trp Thr Arg Asp Ser

           45                   50                  55ggt ctc gtc ctc aag acc ctc gtc gat ctc ttc cga aat gga gat acc       288Gly Leu Val Leu Lys Thr Leu Val Asp Leu Phe Arg Asn Gly Asp Thr45 50 55GGT CTC GTC CTC AAG AAG ACC CTC GTC GATC TTC CGA AAT GGA GAT ACC 288GLY Leu Val Leu Val Asn Gly ARG ASP THR

        60                  65                   70agt ctc ctc tcc acc att gag aac tac atc tcc gcc cag gca att gtc       336Ser Leu Leu Ser Thr Ile Glu Asn Tyr Ile Ser Ala Gln Ala Ile Val60 65 70AGT CTC CTC CTC TCC ACC ATT GAG AAC TAC ATC TCC GCC CAG GCA ATT GTC 336SER Leu Leu

     75                  80                  85cag ggt atc agt aac ccc tct ggt gat ctg tcc agc ggc gct ggt ctc      384Gln Gly Ile Ser Asn Pro Ser Gly Asp Leu Ser Ser Gly Ala Gly Leu75 80 85CAG GGT AGT AGT AAC CCC TCT GGT GGT GAT CTG TCC GGC GGC GCT GGT GGT CTC 384GLN GLY Ile Serite ASP Leu Series Gly Ala Gly Leu

90                  95                  100ggt gaa ccc aag ttc aat gtc gat gag act gcc tac act ggt tct tgg      432Gly Glu Pro Lys Phe Asn Val Asp Glu Thr Ala Tyr Thr Gly Ser Trp105                 110                 115                 120gga cgg ccg cag cga gat ggt ccg gct ctg aga gca act gct atg atc      480Gly Arg Pro Gln Arg Asp Gly Pro Ala Leu Arg Ala Thr Ala Met Ile90                  95                  100ggt gaa ccc aag ttc aat gtc gat gag act gcc tac act ggt tct tgg      432Gly Glu Pro Lys Phe Asn Val Asp Glu Thr Ala Tyr Thr Gly Ser Trp105                 110                 115                 120gga cgg ccg cag cga gat ggt ccg gct ctg aga gca act gct atg atc 480Gly Arg Pro Gln Arg Asp Gly Pro Ala Leu Arg Ala Thr Ala Met Ile

            125                 130                 135ggc ttc ggg cag tgg ctg ctt gac aat ggc tac acc agc acc gca acg      528Gly Phe Gly Gln Trp Leu Leu Asp Asn Gly Tyr Thr Ser Thr Ala Thr            125                 130                 135ggc ttc ggg cag tgg ctg ctt gac aat ggc tac acc agc acc gca acg      528Gly Phe Gly Gln Trp Leu Leu Asp Asn Gly Tyr Thr Ser Thr Ala Thr

        140                 145                 150gac att gtt tgg ccc ctc gtt agg aac gac ctg tcg tat gtg gct caa      576Asp Ile Val Trp Pro Leu Val Arg Asn Asp Leu Ser Tyr Val Ala Gln140 145 150GAC ATT GTT TGG CCC CTC GTT AGG AGG AAC GAC CTG TAT GCT CAA 576ASP Ile Val Tr Leu Val ARG ASER Valr Valr Val Ala Gln

    155                 160                 165tac tgg aac cag aca gga tat gat ctc tgg gaa gaa gtc aat ggc tcg      624Tyr Trp Asn Gln Thr Gly Tyr Asp Leu Trp Glu Glu Val Asn Gly Ser155 160 165TAC TGA CAG ACA GGA TAT GAT GAA GAA GAA GAA GAA GGC TCG 624tyr TRN THR GLY TYR ASP Leu Glu Glu Val asn GLU GLU Val asr Ser

170                 175                 180tct ttc ttt acg att gct gtg caa cac cgc gcc ctt gtc gaa ggt agt      672Ser Phe Phe Thr Ile Ala Val Gln His Arg Ala Leu Val Glu Gly Ser185                 190                 195                 200gcc ttc gcg acg gcc gtc ggc tcg tcc tgc tcc tgg tgt gat tct cag      720Ala Phe Ala Thr Ala Val Gly Ser Ser Cys Ser Trp Cys Asp Ser Gln170                 175                 180tct ttc ttt acg att gct gtg caa cac cgc gcc ctt gtc gaa ggt agt      672Ser Phe Phe Thr Ile Ala Val Gln His Arg Ala Leu Val Glu Gly Ser185                 190                 195                 200gcc ttc gcg acg gcc gtc ggc tcg tcc tgc tcc tgg tgt gat tct cag 720Ala Phe Ala Thr Ala Val Gly Ser Ser Cys Ser Trp Cys Asp Ser Gln

            205                 210                 215gca ccc gaa att ctc tgc tac ctg cag tcc ttc tgg acc ggc agc ttc      768Ala Pro Glu Ile Leu Cys Tyr Leu Gln Ser Phe Trp Thr Gly Ser Phe205 210 215GCA CCC GAA ATT CTC TGC TAC CTG CAG TCC TGG ACC GGC AGC GGC TTC 768ALA GLU Ile Leu Cys Tyr Leu Gln Serp TR GLY Ser Ge Pher GLY SE PHE PHR GLY SE

        220                 225                 230att ctg gcc aac ttc gat agc agc cgt tcc ggc aag gac gca aac acc      816Ile Leu Ala Asn Phe Asp Ser Ser Arg Ser Gly Lys Asp Ala Asn Thr220 225 230ATT CTT CTC AAC TTC GAT AGC AGC CGT TCC GGC AAG GAC GCA ACC 816ile Leu Ala Asn PHE ARG Serg Serg Lysn THRsn Thr

    235                 240                 245ctc ctg gga agc atc cac acc ttt gat cct gag gcc gca tgc gac gac      864Leu Leu Gly Ser Ile His Thr Phe Asp Pro Glu Ala Ala Cys Asp Asp235 240 245CTC CTG GGA AGC AGC ATC CAC ACC TTT GCC GCC GCA TGC GAC GAC 864Leu Leu GLY Serle Hr Pro Glu Ala Ala Cys ASP As

250                 255                 260tcc acc ttc cag ccc tgc tcc ccg cgc gcg ctc gcc aac cac aag gag      912Ser Thr Phe Gln Pro Cys Ser Pro Arg Ala Leu Ala Asn His Lys Glu265                 270                275                  280gtt gta gac tct ttc cgc tca atc tat acc ctc aac gat ggt ctc agt      960Val Val Asp Ser Phe Arg Ser Ile Tyr Thr Leu Asn Asp Gly Leu Ser250                 255                 260tcc acc ttc cag ccc tgc tcc ccg cgc gcg ctc gcc aac cac aag gag      912Ser Thr Phe Gln Pro Cys Ser Pro Arg Ala Leu Ala Asn His Lys Glu265                 270                275                  280gtt gta gac tct ttc cgc tca atc tat acc ctc aac gat ggt ctc agt 960Val Val Asp Ser Phe Arg Ser Ile Tyr Thr Leu Asn Asp Gly Leu Ser

            285                 290                 295gac agc gag gct gtt gcg gtg ggt cgg tac cct gag gac acg tac tac     1008Asp Ser Glu Ala Val Ala Val Gly Arg Tyr Pro Glu Asp Thr Tyr Tyr285 290 295GAC AGC GAG GCT GCG GCG GGT CGG TAC CCT GAC GAC ACG TAC 1008asp Ser Glu Ala Val ARG Tyr Pro Glu ASP ThR Tyr Tyr Tyr Tyr

        300                 305                 310aac ggc aac ccg tgg ttc ctg tgc acc ttg gct gcc gca gag cag ttg     1056Asn Gly Asn Pro Trp Phe Leu Cys Thr Leu Ala Ala Ala Glu Gln Leu300 305 310AAC GGC AAC CCG TGG TGG TTC CTG TGC ACC TTG GCC GCC GCA GAG CAG TTG 1056ASN GLE

    315                 320                 325tac gat gct cta tac cag tgg gac aag cag ggg tcg ttg gag gtc aca     1104Tyr Asp Ala Leu Tyr Gln Trp Asp Lys Gln Gly Ser Leu Glu Val Thr315 320 325TAC GCT GCT CTA TAC CAG GAC AAG GGG GGG TCG TCG GAG GAG GAG GAG GAG GAG GAG GAG GAG GAG GAG GAG GAG GAG GAG GAG GAG GAG

330                 335                 340gat gtg tcg ctg gac ttc ttc aag gca ctg tac agc gat gct gct act     1152Asp Val Ser Leu Asp Phe Phe Lys Ala Leu Tyr Ser Asp Ala Ala Thr345                 350                 355                 360ggc acc tac tct tcg tcc agt tcg act tat agt agc att gta gat gcc     1200Gly Thr Tyr Ser Ser Ser Ser Ser Thr Tyr Ser Ser Ile Val Asp Ala330                 335                 340gat gtg tcg ctg gac ttc ttc aag gca ctg tac agc gat gct gct act     1152Asp Val Ser Leu Asp Phe Phe Lys Ala Leu Tyr Ser Asp Ala Ala Thr345                 350                 355                 360ggc acc tac tct tcg tcc agt tcg act tat agt agc att gta gat gcc 1200Gly Thr Tyr Ser Ser Ser Ser Ser Ser Thr Tyr Ser Ser Ile Val Asp Ala

            365                 370                 375gtg aag at ttc gcc gat ggc ttc gtc tct att gtg gaa act cac gcc     1248Val Lys Thr Phe Ala Asp Gly Phe Val Ser Ile Val Glu Thr His Ala365 370 375GTG Aag at TTC GCC GCC GGC TTC GTC TCT ATT GAA ACT CAC GCC 1248VAL LYS Thr PHE ALA ALA ASP GLE VAL GLU Thr His Ala

        380                 385                  390gca agc aac ggc tcc atg tcc gag caa tac gac aag tct gat ggc gag     1296Ala Ser Asn Gly Ser Met Ser Glu Gln Tyr Asp Lys Ser Asp Gly Glu380 385 390GCA AGC AAC GGC TCC ATG TCC GAG CAA TAC GAC AAG TCT GGC GGC GAG 1296ALA Sern Gly Sergial Sergrn Tyr ASP GLY GLY GLY GLY GLY GLY GLY GLY

    395                 400                 405cag ctt tcc gct cgc gac ctg acc tgg tct tat gct gct ctg ctg acc     1344Gln Leu Ser Ala Arg Asp Leu Thr Trp Ser Tyr Ala Ala Leu Leu Thr395 4005CAG CTT TCC GCT CGC GAC CTG ACC TGG TAT GCT GCT GCT GCT CTG ACC 1344GLN Leu Serg ARG ASP Leu ThR Tyr Ala Ala Leu Leu Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr ThR

410                 415                 420gcc aac aac cgt cgt aac tcc gtc gtg cct gct tct tgg ggc gag acc     1392Ala Asn Asn Arg Arg Asn Ser Val Val Pro Ala Ser Trp Gly Glu Thr425                 430                 435                 440tct gcc agc agc gtg ccc ggc acc tgt gcg gcc aca tct gcc att ggt     1440Ser Ala Ser Ser Val Pro Gly Thr Cys Ala Ala Thr Ser Ala Ile Gly410                 415                 420gcc aac aac cgt cgt aac tcc gtc gtg cct gct tct tgg ggc gag acc     1392Ala Asn Asn Arg Arg Asn Ser Val Val Pro Ala Ser Trp Gly Glu Thr425                 430                 435                 440tct gcc agc agc gtg ccc ggc acc tgt gcg gcc aca tct gcc att ggt 1440Ser Ala Ser Ser Ser Val Pro Gly Thr Cys Ala Ala Thr Ser Ala Ile Gly

            445                 450                 455acc tac agc agt gtg act gtc acc tcg tgg ccg agt atc gtg gct act     1488Thr Tyr Ser Ser Val Thr Val Thr Ser Trp Pro Ser Ile Val Ala Thr445 450 455ACC TAC AGC AGT GTG ACT GTC ACC TCG TGG CCG AGT ATC GCT Act 1488thr Tyr Ser Val THR VR Val TRALA's

        460                 465                 470ggc ggc acc act acg acg gct acc ccc act gga tcc ggc agc gtg acc     1536Gly Gly Thr Thr Thr Thr Ala Thr Pro Thr Gly Ser Gly Ser Val Thr460 465 470GC GGC ACC ACG ACG ACG GCT ACC CCC ACC GGC GGC GGC GGC GTG ACC 1536gly GLY ThR THR ALA THR GLY Ser Val Ser Val THR THR THR THR THR THR THR THR THR Val THR THR Val THR THR Val THR Val Thr Val Thr Val Thr Val THR Val THR TH -THR that

    475                 480                 485tcg acc agc aag acc acc gcg act gct agc aag acc agc acc acg acc     1584Ser Thr Ser Lys Thr Thr Ala Thr Ala Ser Lys Thr Ser Thr Thr Thr    475                 480                 485tcg acc agc aag acc acc gcg act gct agc aag acc agc acc acg acc     1584Ser Thr Ser Lys Thr Thr Ala Thr Ala Ser Lys Thr Ser Thr Thr Thr

490                 495                 500cgc tct ggt atg tca ctg tga                                         1605Arg Ser Gly Met Ser Leu505                 510<210>2<211>534<212>PRT<213>黑曲霉(Aspergillus niger)<220><221>signal<222>(1)…(24)<400>2Met Ser Phe Arg Ser Leu Leu Ala Leu Ser Gly Leu Val Cys Thr Gly490 495 500CGC TCT GGT ATG TCA CTG TGA 1605ARG Ser Gly Met Serou505 510 <210> 2 <211> 534 <212> PRT <213> Aspergillus niger <220> <221> Signa <222> (1) …(24)<400>2Met Ser Phe Arg Ser Leu Leu Ala Leu Ser Gly Leu Val Cys Thr Gly

            -20                 -15                 -10Leu Ala Asn Val Ile Ser Lys Arg Ala Thr Leu Asp Ser Trp Leu Ser-20 -15 -10Leu Ala Asn Val Ile Ser Lys Arg Ala Thr Leu Asp Ser Trp Leu Ser

        -5                   1               5Asn Glu Ala Thr Val Ala Arg Thr Ala Ile Leu Asn Asn Ile Gly Ala-5 1 1 5Asn Glu Ala Thr Val Ala Arg Thr Ala Ile Leu Asn Asn Ile Gly Ala

10                  15                  20Asp Gly Ala Trp Val Ser Gly Ala Asp Ser Gly Ile Val Val Ala Ser25                  30                  35                  40Pro Ser Thr Asp Asn Pro Asp Tyr Phe Tyr Thr Trp Thr Arg Asp Ser10 15 20ASP GLY ALA TRP Val Serg Ala aser Gly Ile Val Val Ala Ser25 30 35 40pro Ser THR ASP TYR PHR THR THR THR ARG ASP Serg ARG ASP Serg ARG ASP Serg ARG ASP SE

            45                  50                   55Gly Leu Val Leu Lys Thr Leu Val Asp Leu Phe Arg Asn Gly Asp Thr45 50 55Gly Leu Val Leu Lys Thr Leu Val Asp Leu Phe Arg Asn Gly Asp Thr

        60                  65                  70Ser Leu Leu Ser Thr Ile Glu Asn Tyr Ile Ser Ala Gln Ala Ile Val60 65 70Ser Leu Leu Ser Thr Ile Glu Asn Tyr Ile Ser Ala Gln Ala Ile Val

    75                  80                  85Gln Gly Ile Ser Asn Pro Ser Gly Asp Leu Ser Ser Gly Ala Gly Leu75 80 85Gln Gly Ile Ser Asn Pro Ser Gly Asp Leu Ser Ser Gly Ala Gly Leu

90                  95                  100Gly Glu Pro Lys Phe Asn Val Asp Glu Thr Ala Tyr Thr Gly Ser Trp105                 110                 115                 120Gly Arg Pro Gln Arg Asp Gly Pro Ala Leu Arg Ala Thr Ala Met Ile90 95 100Gly GLU Pro Lys PHE Asn Val ASP GLU THR Ala Tyr THR GLY Serp105 110 115 120GLY ARG Pro Gln ARG Ala Ala THR ALA MET Ile

            125                 130                 135Gly Phe Gly Gln Trp Leu Leu Asp Asn Gly Tyr Thr Ser Thr Ala Thr125 130 135Gly Phe Gly Gln Trp Leu Leu Asp Asn Gly Tyr Thr Ser Thr Ala Thr

        140                 145                 150Asp Ile Val Trp Pro Leu Val Arg Asn Asp Leu Ser Tyr Val Ala Gln140 145 150Asp Ile Val Trp Pro Leu Val Arg Asn Asp Leu Ser Tyr Val Ala Gln

    155                 160                 165Tyr Trp Asn Gln Thr Gly Tyr Asp Leu Trp Glu Glu Val Asn Gly Ser155 160 165Tyr Trp Asn Gln Thr Gly Tyr Asp Leu Trp Glu Glu Val Asn Gly Ser

170                 175                 180Ser Phe Phe Thr Ile Ala Val Gln His Arg Ala Leu Val Glu Gly Ser185                 190                 195                 200Ala Phe Ala Thr Ala Val Gly Ser Ser Cys Ser Trp Cys Asp Ser Gln170 175 180r PHE PHE Thr Ile Ala Val Gln His ARG Ala Leu Val GLU GLY Ser185 195 200ALA PHE ALA THR Ala Val Ser Ser Trp Cys ASP Ser Gln Gln Gln Gln Gln Gln Gln Gln

            205                 210                 215Ala Pro Glu Ile Leu Cys Tyr Leu Gln Ser Phe Trp Thr Gly Ser Phe205 210 215Ala Pro Glu Ile Leu Cys Tyr Leu Gln Ser Phe Trp Thr Gly Ser Phe

       220                  225                 230Ile Leu Ala Asn Phe Asp Ser Ser Arg Ser Gly Lys Asp Ala Asn Thr220 225 230Ile Leu Ala Asn Phe Asp Ser Ser Arg Ser Gly Lys Asp Ala Asn Thr

    235                 240                 245Leu Leu Gly Ser Ile His Thr Phe Asp Pro Glu Ala Ala Cys Asp Asp235 240 245Leu Leu Gly Ser Ile His Thr Phe Asp Pro Glu Ala Ala Cys Asp Asp

250                 255                 260Ser Thr Phe Gln Pro Cys Ser Pro Arg Ala Leu Ala Asn His Lys Glu265                 270                 275                 280Val Val Asp Ser Phe Arg Ser Ile Tyr Thr Leu Asn Asp Gly Leu Ser250 255 260SER Thr Phe Gln Pro Cys Serg Ala Leu Ala Asn His Lys GLU265 275 280VAL VAL ARG Ser, Ile Tyr Leu asn Asn Asn ASP GLY Leu Ser

            285                 290                 295Asp Ser Glu Ala Val Ala Val Gly Arg Tyr Pro Glu Asp Thr Tyr Tyr285 290 295Asp Ser Glu Ala Val Ala Val Gly Arg Tyr Pro Glu Asp Thr Tyr Tyr

        300                 305                 310Asn Gly Asn Pro Trp Phe Leu Cys Thr Leu Ala Ala Ala Glu Gln Leu300 305 310Asn Gly Asn Pro Trp Phe Leu Cys Thr Leu Ala Ala Ala Glu Gln Leu

    315                 320                 325Tyr Asp Ala Leu Tyr Gln Trp Asp Lys Gln Gly Ser Leu Glu Val Thr315 320 325Tyr Asp Ala Leu Tyr Gln Trp Asp Lys Gln Gly Ser Leu Glu Val Thr

330                 335                 340Asp Val Ser Leu Asp Phe Phe Lys Ala Leu Tyr Ser Asp Ala Ala Thr345                 350                 355                 360Gly Thr Tyr Ser Ser Ser Ser Ser Thr Tyr Ser Ser Ile Val Asp Ala330 335 340ASP VASP PHE PHE PHE PHE PHE PHE LYS ALA Leu Tyr Seru Ala Ala ThR345 350 360GLY Thr Ser Ser Ser Ser Seer Val Ile Val ALA

            365                 370                 375Val Lys Thr Phe Ala Asp Gly Phe Val Ser Ile Val Glu Thr His Ala365 370 375Val Lys Thr Phe Ala Asp Gly Phe Val Ser Ile Val Glu Thr His Ala

        380                 385                 390Ala Ser Asn Gly Ser Met Ser Glu Gln Tyr Asp Lys Ser Asp Gly Glu380 385 390Ala Ser Asn Gly Ser Met Ser Glu Gln Tyr Asp Lys Ser Asp Gly Glu

    395                 400                 405Gln Leu Ser Ala Arg Asp Leu Thr Trp Ser Tyr Ala Ala Leu Leu Thr395 400 405Gln Leu Ser Ala Arg Asp Leu Thr Trp Ser Tyr Ala Ala Leu Leu Thr

410                 415                 420Ala Asn Asn Arg Arg Asn Ser Val Val Pro Ala Ser Trp Gly Glu Thr425                 430                 435                 440Ser Ala Sar Ser Val Pro Gly Thr Cys Ala Ala Thr Ser Ala Ile Gly410 415 420ALA Asn ARG ARG ARG ASN Ser Val Val Val Pro Ala Serp Gly GLU ThR425 435 440r Sar Val Pro Gly Thr Cys Ala THRA Ile Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly

            445                 450                 455Thr Tyr Ser Ser Val Thr Val Thr Ser Trp Pro Ser Ile Val Ala Thr445 450 455Thr Tyr Ser Ser Val Thr Val Thr Ser Trp Pro Ser Ile Val Ala Thr

        460                 465                 470Gly Gly Thr Thr Thr Thr Ala Thr Pro Thr Gly Ser Gly Ser Val Thr460 465 470Gly Gly Thr Thr Thr Thr Ala Thr Pro Thr Gly Ser Gly Ser Val Thr

    475                 480                 485Ser Thr Ser Lys Thr Thr Ala Thr Ala Ser Lys Thr Ser Thr Thr Thr475 480 485Ser Thr Ser Lys Thr Thr Ala Thr Ala Ser Lys Thr Ser Thr Thr Thr Thr

490                 495                 500Arg Ser Gly Met Ser Leu505                 510490 495 500Arg Ser Gly Met Ser Leu505 510

<210>3<210>3

<211>30<211>30

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<220><220>

<223>引物7258<223> Primer 7258

<400>3gaatgacttg gttgacgcgt caccagtcac                                     30<400>3gaatgacttg gttgacgcgt caccagtcac 30

<210>4<210>4

<211>68<211>68

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<220><220>

<223>引物21401<223> Primer 21401

<400>4ggggatcatg ataggactag ccatattaat gaagggcata taccacgcct tggacctgcg    60ttatagcc                                                             68<400>4ggggatcatg ataggactag ccatattaat gaagggcata taccacgcct tggacctgcg 60ttatagcc 68

<210>5<210>5

<211>25<211>25

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<220><220>

<223>引物107581<223> Primer 107581

<400>5gcaacgaagc gcccgtggct cgtac                                          25<400>5gcaacgaagc gcccgtggct cgtac 25

<210>6<210>6

<211>88<211>88

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<220><220>

<223>引物FAMGII<223> Primer FAMGII

<400>6cgaagcgacc gtggctcgta ctgccatcta taacatcggc gcgtctgtgc gcggtggcat    60tgtcgttgct agtcccagca cggataac                                       88<400>6cgaagcgacc gtggctcgta ctgccatcta taacatcggc gcgtctgtgc gcggtggcat 60tgtcgttgct agtcccagca cggataac 88

<210>7<210>7

<211>28<211>28

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<220><220>

<223>引物RAMG1<223> Primer RAMG1

<400>7gatggcagta cgagccacgg tcgcttcg                                       28<400>7gatggcagta cgagccacgg tcgcttcg 28

<210>8<210>8

<211>75<211>75

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<220><220>

<223>引物FAMGIV<223> Primer FAMGIV

<400>8gtgtcgctgg acttcttcaa gaacctctta ccctactaca gtcgttatca ttgatgccgt    60gaagactttc gccga                                                     75<400>8gtgtcgctgg acttcttcaa gaacctctta ccctactaca gtcgttatca ttgatgccgt 60gaagactttc gccga 75

<210>9<210>9

<211>21<211>21

<212>DNA<212>DNA

<213>引物RAMGVI<213> Primer RAMGVI

<400>9cttgaagaag tccagcgaca c                                              21<400>9cttgaagaag tccagcgaca c 21

<210>10<210>10

<211>27<211>27

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<220><220>

<223>引物FG2<223> Primer FG2

<400>10catccccagg atccttactc agcaatg                                         27<400>10catccccagg atccttactc agcaatg 27

<210>11<210>11

<211>27<211>27

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<220><220>

<223>引物RG2<223> Primer RG2

<400>11ctcaaacgac tcaccagcct ctagagt                                          27<400>11ctcaaacgac tcaccagcct ctagagt 27

<210>12<210>12

<211>2602<211>2602

<212>DNA<212>DNA

<213>黑曲霉(ASPERGILLUS NIGER)<213> Aspergillus niger (ASPERGILLUS NIGER)

<400>12ttcgtcgcct aatgtctcgt ccgttcacaa actgaagagc ttgaagtggc gagatgtctc      60tgcaggaatt caagctagat gctaagcgat attgcatggc aatatgtgtt gatgcatgtg     120cttcttcctt cagcttcccc tcgtgcgagt gaggtttggc tataaattga agtggttggt     180cggggttccg tgaggggctg aagtgcttcc tcccttttag gcgcaactga gagcctgagc     240ttcatcccca gcatcattac acctcagcaa tgtcgttccg atctctactc gccctgagcg     300gcctcgtctg cacagggttg gcaaatgtga tttccaagcg cgcgaccttg gattcatggt     360tgagcaacga agcgaccgtg gctcgtactg ccatcctgaa taacatcggg gcggacggtg     420cttgggtgtc gggcgcggac tctggcattg tcgttgctag tcccagcacg gataacccgg     480actgtatgtt tcgagctcag atttagtatg agtgtgtcat tgattgattg atgctgactg     540gcgtgtcgtt tgttgtagac ttctacacct ggactcgcga ctctggtctc gtcctcaaga     600ccctcgtcga tctcttccga aatggagata ccagtctcct ctccaccatt gagaactaca     660tctccgccca ggcaattgtc cagggtatca gtaacccctc tggtgatctg tccagcggcg     720ctggtctcgg tgaacccaag ttcaatgtcg atgagactgc ctacactggt tcttggggac     780ggccgcagcg agatggtccg gctctgagag caactgctat gatcggcttc gggcagtggc     840tgcttgtatg ttctccaccc ccttgcgtct gatctgtgac atatgtagct gactggtcag     900gacaatggct acaccagcac cgcaacggac attgtttggc ccctcgttag gaacgacctg     960tcgtatgtgg ctcaatactg gaaccagaca ggatatggtg tgtttgtttt attttaaatt    1020tccaaagatg cgccagcaga gctaacccgc gatcgcagat ctctgggaag aagtcaatgg    1080ctcgtctttc tttacgattg ctgtgcaaca ccgcgccctt gtcgaaggta gtgccttcgc    1140gacggccgtc ggctcgtcct gctcctggtg tgattctcag gcacccgaaa ttctctgcta    1200cctgcagtcc ttctggaccg gcagcttcat tctggccaac ttcgatagca gccgttccgg    1260caaggacgca aacaccctcc tgggaagcat ccacaccttt gatcctgagg ccgcatgcga    1320cgactccacc ttccagccct gctccccgcg cgcgctcgcc aaccacaagg aggttgtaga    1380ctctttccgc tcaatctata ccctcaacga tggtctcagt gacagcgagg ctgttgcggt    1440gggtcggtac cctgaggaca cgtactacaa cggcaacccg tggttcctgt gcaccttggc    1500tgccgcagag cagttgtacg atgctctata ccagtgggac aagcaggggt cgttggaggt    1560cacagatgtg tcgctggact tcttcaaggc actgtacagc gatgctgcta ctggcaccta    1620ctcttcgtcc agttcgactt atagtagcat tgtagatgcc gtgaagactt tcgccgatgg    1680cttcgtctct attgtggtaa gtctacgcta gacaagcgct catgttgaca gagggtgcgt    1740actaacagaa gtaggaaact cacgccgcaa gcaacggctc catgtccgag caatacgaca    1800agtctgatgg cgagcagctt tccgctcgcg acctgacctg gtcttatgct gctctgctga    1860ccgccaacaa ccgtcgtaac tccgtcgtgc ctgcttcttg gggcgagacc tctgccagca    1920gcgtgcccgg cacctgtgcg gccacatctg ccattggtac ctacagcagt gtgactgtca    1980cctcgtggcc gagtatcgtg gctactggcg gcaccactac gacggctacc cccactggat    2040ccggcagcgt gacctcgacc agcaagacca ccgcgactgc tagcaagacc agcaccagta    2100cgtcatcaac ctcctgtacc actcccaccg ccgtggctgt gactttcgat ctgacagcta    2160ccaccaccta cggcgagaac atctacctgg tcggatcgat ctctcagctg ggtgactggg    2220aaaccagcga cggcatagct ctgagtgctg acaagtacac ttccagcgac ccgctctggt    2280atgtcactgt gactctgccg gctggtgagt cgtttgagta caagtttatc cgcattgaga    2340gcgatgactc cgtggagtgg gagagtgatc ccaaccgaga atacaccgtt cctcaggcgt    2400gcggaacgtc gaccgcgacg gtgactgaca cctggcggtg acaatcaatc catttcgcta    2460tagttaaagg atggggatga gggcaattgg ttatatgatc atgtatgtag tgggtgtgca    2520taatagtagt gaaatggaag ccaagtcatg tgattgtaat cgaccgacgg aattgaggat    2580atccggaaat acagacaccg gg                                              2602<400>12ttcgtcgcct aatgtctcgt ccgttcacaa actgaagagc ttgaagtggc gagatgtctc      60tgcaggaatt caagctagat gctaagcgat attgcatggc aatatgtgtt gatgcatgtg     120cttcttcctt cagcttcccc tcgtgcgagt gaggtttggc tataaattga agtggttggt     180cggggttccg tgaggggctg aagtgcttcc tcccttttag gcgcaactga gagcctgagc     240ttcatcccca gcatcattac acctcagcaa tgtcgttccg atctctactc gccctgagcg     300gcctcgtctg cacagggttg gcaaatgtga tttccaagcg cgcgaccttg gattcatggt     360tgagcaacga agcgaccgtg gctcgtactg ccatcctgaa taacatcggg gcggacggtg     420cttgggtgtc gggcgcggac tctggcattg tcgttgctag tcccagcacg gataacccgg     480actgtatgtt tcgagctcag atttagtatg agtgtgtcat tgattgattg atgctgactg     540gcgtgtcgtt tgttgtagac ttctacacct ggactcgcga ctctggtctc gtcctcaaga     600ccctcgtcga tctcttccga aatggagata ccagtctcct ctccaccatt gagaactaca     660tctccgccca ggcaattgtc cagggtatca gtaacccctc tggtgatctg tccagcggcg     720ctggtctcgg tgaacccaag ttcaatgtcg atgagactgc ctacactggt tcttggggac     780ggccgcagcg agatggtccg gctctgagag caactgctat gatcggcttc gggcagtggc     840tgcttgtatg ttctccaccc ccttgcgtct gatctgtgac atatgtagct gactggtcag     900gacaatggct acaccagcac cgcaacggac attgtttggc ccctcgttag gaacgacctg     960tcgtatgtgg ctcaatactg gaaccagaca ggatatggtg tgtttgtttt attttaaatt    1020tccaaagatg cgccagcaga gctaacccgc gatcgcagat ctctgggaag aagtcaatgg    1080ctcgtctttc tttacgattg ctgtgcaaca ccgcgccctt gtcgaaggta gtgccttcgc    1140gacggccgtc ggctcgtcct gctcctggtg tgattctcag gcacccgaaa ttctctgcta    1200cctgcagtcc ttctggaccg gcagcttcat tctggccaac ttcgatagca gccgttccgg    1260caaggacgca aacaccctcc tgggaagcat ccacaccttt gatcctgagg ccgcatgcga    1320cgactccacc ttccagccct gctccccgcg cgcgctcgcc aaccacaagg aggttgtaga    1380ctctttccgc tcaatctata ccctcaacga tggtctcagt gacagcgagg ctgttgcggt    1440gggtcggtac cctgaggaca cgtactacaa cggcaacccg tggttcctgt gcaccttggc    1500tgccgcagag cagttgtacg atgctctata ccagtgggac aagcaggggt cgttggaggt    1560cacagatgtg tcgctggact tcttcaaggc actgtacagc gatgctgcta ctggcaccta    1620ctcttcgtcc agttcgactt atagtagcat tgtagatgcc gtgaagactt tcgccgatgg    1680cttcgtctct attgtggtaa gtctacgcta gacaagcgct catgttgaca gagggtgcgt    1740actaacagaa gtaggaaact cacgccgcaa gcaacggctc catgtccgag caatacgaca    1800agtctgatgg cgagcagctt tccgctcgcg acctgacctg gtcttatgct gctctgctga    1860ccgccaacaa ccgtcgtaac tccgtcgtgc ctgcttcttg gggcgagacc tctgccagca    1920gcgtgcccgg cacctgtgcg gccacatctg ccattggtac ctacagcagt gtgactgtca    1980cctcgtggcc gagtatcgtg gctactggcg gcaccactac gacggctacc cccactggat    2040ccggcagcgt gacctcgacc agcaagacca ccgcgactgc tagcaagacc agcaccagta    2100cgtcatcaac ctcctgtacc actcccaccg ccgtggctgt gactttcgat ctgacagcta    2160ccaccaccta cggcgagaac atctacctgg tcggatcgat ctctcagctg ggtgactggg    2220aaaccagcga cggcatagct ctgagtgctg acaagtacac ttccagcgac ccgctctggt    2280atgtcactgt gactctgccg gctggtgagt cgtttgagta caagtttatc cgcattgaga    2340gcgatgactc cgtggagtgg gagagtgatc ccaaccgaga atacaccgtt cctcaggcgt    2400gcggaacgtc gaccgcgacg gtgactgaca cctggcggtg acaatcaatc catttcgcta    2460tagttaaagg Atggggatga GGGCCGG TTATATGATGATGTAG TGGGGTGCA 2520TAATATGT GAAAAGGAGAAGGAGAAGGAAGTCATGTCATGTGACGACGAGGGAT 2580ATCCGGACACACCG GG 260260260

<210> 13<210> 13

<211> 640<211> 640

<212> PRT<212> PRT

<213> 黑曲霉(ASPERGILLUS NIGER)<213> Aspergillus niger (ASPERGILLUS NIGER)

<400> 13Met Ser Phe Arg Ser Leu Leu Ala Leu Ser Gly Leu Val Cys Thr Gly1               5                  10                  15Leu Ala Asn Val Ile Ser Lys Arg Ala Thr Leu Asp Ser Trp Leu Ser<400> 13MET Serg Serg Serite

        20                  25                   30Asn Glu Ala Thr Val Ala Arg Thr Ala Ile Leu Asn Asn Ile Gly Ala20 25 25 30Asn Glu Ala Thr Val Ala Arg Thr Ala Ile Leu Asn Asn Ile Gly Ala

    35                  40                  45Asp Gly Ala Trp Val Ser Gly Ala Asp Ser Gly Ile Val Val Ala Ser35 40 45Asp Gly Ala Trp Val Ser Gly Ala Asp Ser Gly Ile Val Val Ala Ser

50                  55                  60Pro Ser Thr Asp Asn Pro Asp Tyr Phe Tyr Thr Trp Thr Arg Asp Ser65                  70                  75                   80Gly Leu Val Leu Lys Thr Leu Val Asp Leu Phe Arg Asn Gly Asp Thr50 55 60pro Ser THR ASP Asn Pro ASP TYR PHE TYR THR THR ARG ASP Ser65 70 75 80Gly Leu Val THR Leu Val Asn Gly ASP THR

            85                  90                  95Ser Leu Leu Ser Thr Ile Glu Asn Tyr Ile Ser Ala Gln Ala Ile Val85 90 95Ser Leu Leu Ser Thr Ile Glu Asn Tyr Ile Ser Ala Gln Ala Ile Val

       100                 105                  110Gln Gly Ile Ser Asn Pro Ser Gly Asp Leu Ser Ser Gly Ala Gly Leu100 105 110Gln Gly Ile Ser Asn Pro Ser Gly Asp Leu Ser Ser Gly Ala Gly Leu

    115                 120                 125Gly Glu Pro Lys Phe Asn Val Asp Glu Thr Ala Tyr Thr Gly Ser Trp115 120 125Gly Glu Pro Lys Phe Asn Val Asp Glu Thr Ala Tyr Thr Gly Ser Trp

130                 135                 140Gly Arg Pro Gln Arg Asp Gly Pro Ala Leu Arg Ala Thr Ala Met Ile145                150                 155                  160Gly Phe Gly Gln Trp Leu Leu Asp Asn Gly Tyr Thr Ser Thr Ala Thr130                 135                 140Gly Arg Pro Gln Arg Asp Gly Pro Ala Leu Arg Ala Thr Ala Met Ile145                150                 155                  160Gly Phe Gly Gln Trp Leu Leu Asp Asn Gly Tyr Thr Ser Thr Ala Thr

            165                 170                 175Asp Ile Val Trp Pro Leu Val Arg Asn Asp Leu Ser Tyr Val Ala Gln165 170 175Asp Ile Val Trp Pro Leu Val Arg Asn Asp Leu Ser Tyr Val Ala Gln

        180                 185                190Tyr Trp Asn Gln Thr Gly Tyr Asp Leu Trp Glu Glu Val Asn Gly Ser180 185 190Tyr Trp Asn Gln Thr Gly Tyr Asp Leu Trp Glu Glu Val Asn Gly Ser

    195                 200                 205Ser Phe Phe Thr Ile Ala Val Gln His Arg Ala Leu Val Glu Gly Ser195 200 205 Ser Phe Phe Thr Ile Ala Val Gln His Arg Ala Leu Val Glu Gly Ser

210                 215                 220Ala Phe Ala Thr Ala Val Gly Ser Ser Cys Ser Trp Cys Asp Ser Gln225                 230                 235                 240Ala Pro Glu Ile Leu Cys Tyr Leu Gln Ser Phe Trp Thr Gly Ser Phe210 215 220ALA PHR THR Ala Val Gly SER CYS SER TRP CYS ASP Sergln225 235 240ALA PRO GLU ILE Leu Gln Seru Gln Serp THR GLY Ser Phe

            245                 250                 255Ile Leu Ala Asn Phe Asp Ser Ser Arg Ser Gly Lys Asp Ala Asn Thr245 250 255Ile Leu Ala Asn Phe Asp Ser Ser Arg Ser Gly Lys Asp Ala Asn Thr

        260                 265                 270Leu Leu Gly Ser Ile His Thr Phe Asp Pro Glu Ala Ala Cys Asp Asp260 265 270Leu Leu Gly Ser Ile His Thr Phe Asp Pro Glu Ala Ala Cys Asp Asp

    275                 280                 285Ser Thr Phe Gln Pro Cys Ser Pro Arg Ala Leu Ala Asn His Lys Glu275 280 285Ser Thr Phe Gln Pro Cys Ser Pro Arg Ala Leu Ala Asn His Lys Glu

290                 295                 300Val Val Asp Ser Phe Arg Ser Ile Tyr Thr Leu Asn Asp Gly Leu Ser305                 310                 315                 320Asp Ser Glu Ala Val Ala Val Gly Arg Tyr Pro Glu Asp Thr Tyr Tyr290 295 300Val Val ASP Serg Serg Serite Tyr Tyr Leu asn Asp Gly Leu Ser305 315 320ASP Ser Glu Val Ala Val Gly ARG Tyr Pro Glu ASP ThR Tyr Tyr Tyr.

            325                 330                 335Asn Gly Asn Pro Trp Phe Leu Cys Thr Leu Ala Ala Ala Glu Gln Leu325 330 335Asn Gly Asn Pro Trp Phe Leu Cys Thr Leu Ala Ala Ala Glu Gln Leu

        340                 345                 350Tyr Asp Ala Leu Tyr Gln Trp Asp Lys Gln Gly Ser Leu Glu Val Thr340 345 350Tyr Asp Ala Leu Tyr Gln Trp Asp Lys Gln Gly Ser Leu Glu Val Thr

    355                 360                 365Asp Val Ser Leu Asp Phe Phe Lys Ala Leu Tyr Ser Asp Ala Ala Thr355 360 365Asp Val Ser Leu Asp Phe Phe Lys Ala Leu Tyr Ser Asp Ala Ala Thr

370                 375                 380Gly Thr Tyr Ser Ser Ser Ser Ser Thr Tyr Ser Ser Ile Val Asp Ala385                 390                 395                 400Val Lys Thr Phe Ala Asp Gly Phe Val Ser Ile Val Glu Thr His Ala370 375 380gly Thr Tyr Ser Ser Ser Ser Sering Val Ile Val ALA385 395 400VAL LYS ThR PHE ASP GLY PHE VAL VAL GLU THR His Ala

            405                 410                 415Ala Ser Asn Gly Ser Met Ser Glu Gln Tyr Asp Lys Ser Asp Gly Glu405 410 415Ala Ser Asn Gly Ser Met Ser Glu Gln Tyr Asp Lys Ser Asp Gly Glu

        420                 425                 430Gln Leu Ser Ala Arg Asp Leu Thr Trp Ser Tyr Ala Ala Leu Leu Thr420 425 430Gln Leu Ser Ala Arg Asp Leu Thr Trp Ser Tyr Ala Ala Leu Leu Thr

    435                 440                 445Ala Asn Asn Arg Arg Asn Ser Val Val Pro Ala Ser Trp Gly Glu Thr435 440 445Ala Asn Asn Arg Arg Asn Ser Val Val Pro Ala Ser Trp Gly Glu Thr

450                 455                 460Ser Ala Ser Ser Val Pro Gly Thr Cys Ala Ala Thr Ser Ala Ile Gly465                 470                 475                 480Thr Tyr Ser Ser Val Thr Val Thr Ser Trp Pro Ser Ile Val Ala Thr450                 455                 460Ser Ala Ser Ser Val Pro Gly Thr Cys Ala Ala Thr Ser Ala Ile Gly465                 470                 475                 480Thr Tyr Ser Ser Val Thr Val Thr Ser Trp Pro Ser Ile Val Ala Thr

            485                 490                 495Gly Gly Thr Thr Thr Thr Ala Thr Pro Thr Gly Ser Gly Ser Val Thr485 490 495Gly Gly Thr Thr Thr Thr Ala Thr Pro Thr Gly Ser Gly Ser Val Thr

        500                 505                 510Ser Thr Ser Lys Thr Thr Ala Thr Ala Ser Lys Thr Ser Thr Ser Thr500 505 510Ser Thr Ser Lys Thr Thr Ala Thr Ala Ser Lys Thr Ser Thr Ser Thr

    515                 520                 525Ser Ser Thr Ser Cys Thr Thr Pro Thr Ala Val Ala Val Thr Phe Asp515 520 525Ser Ser Thr Ser Cys Thr Thr Pro Thr Ala Val Ala Val Thr Phe Asp

530                 535                 540Leu Thr Ala Thr Thr Thr Tyr Gly Glu Asn Ile Tyr Leu Val Gly Ser545                 550                 555                 560Ile Ser Gln Leu Gly Asp Trp Glu Thr Ser Asp Gly Ile Ala Leu Ser530 535 540leu Thr Ala THR THR TYR TYR GLY GLU Asn Ile Tyr Leu Val Gly Ser545 550 560ile Seru Gln Leu Glu Thr Serle Ala Leu Ser

            565                 570                 575Ala Asp Lys Tyr Thr Ser Ser Asp Pro Leu Trp Tyr Val Thr Val Thr565 570 575Ala Asp Lys Tyr Thr Ser Ser Asp Pro Leu Trp Tyr Val Thr Val Thr

        580                 585                 590Leu Pro Ala Gly Glu Ser Phe Glu Tyr Lys Phe Ile Arg Ile Glu Ser580 585 590Leu Pro Ala Gly Glu Ser Phe Glu Tyr Lys Phe Ile Arg Ile Glu Ser

    595                 600                 605Asp Asp Ser Val Glu Trp Glu Ser Asp Pro Asn Arg Glu Tyr Thr Val595 600 605Asp Asp Ser Val Glu Trp Glu Ser Asp Pro Asn Arg Glu Tyr Thr Val

610                 615                 620Pro Gln Ala Cys Gly Thr Ser Thr Ala Thr Val Thr Asp Thr Trp Arg625                 630                 635                 640610 615 620Pro Gln Ala Cys Gly Thr Sera Ala THR VR ASP TRP ARG625 630 635 640

Claims (30)

1.亲本葡糖淀粉酶的变体，其包含在下列一或多个位置的改变：59，66，72，119，189，223，227，313，340，342，352，379，386，393，395，402，408，416，425，427，444，486，490，494，其中(a)所述每一改变是1. A variant of the parent glucoamylase comprising an alteration at one or more of the following positions: 59, 66, 72, 119, 189, 223, 227, 313, 340, 342, 352, 379, 386, 393 , 395, 402, 408, 416, 425, 427, 444, 486, 490, 494, wherein (a) each change is (i)在占据此位置的氨基酸下游插入氨基酸，(i) an amino acid is inserted downstream of the amino acid occupying that position, (ii)占据此位置的氨基酸的缺失，或(ii) a deletion of the amino acid occupying this position, or (iii)占据此位置的氨基酸用另一氨基酸取代，(iii) the amino acid occupying this position is substituted with another amino acid, (b)所述变体具有葡糖淀粉酶活性且(c)每一位置对应于具有SEQ IDNO：2之氨基酸序列的亲本葡糖淀粉酶氨基酸序列的位置。(b) the variant has glucoamylase activity and (c) each position corresponds to the position of the parent glucoamylase amino acid sequence having the amino acid sequence of SEQ ID NO:2. 2.亲本葡糖淀粉酶的变体，其包含下列的一或多个改变：V59A，L66V/R，T72I，S119P，I189T，Y223F，F227Y，N313G，S340G，K352R，S356G，T379A，S386K，N，R，P，A393R，S395R，Y402F，E408R，T416A，R，D，N，C，Q，G，H，I，L，K，M，F，P，S，E，W，Y，V，优选T416H，A425T，N427S/M，S444G，S486G，T490A，T494P/A，其中(a)所述变体具有葡糖淀粉酶活性且(b)每一位置对应于具有SEQ ID NO：2之氨基酸序列的亲本葡糖淀粉酶氨基酸序列的位置。2. A variant of the parent glucoamylase comprising one or more of the following changes: V59A, L66V/R, T72I, S119P, I189T, Y223F, F227Y, N313G, S340G, K352R, S356G, T379A, S386K, N , R, P, A393R, S395R, Y402F, E408R, T416A, R, D, N, C, Q, G, H, I, L, K, M, F, P, S, E, W, Y, V , preferably T416H, A425T, N427S/M, S444G, S486G, T490A, T494P/A, wherein (a) the variant has glucoamylase activity and (b) each position corresponds to a sequence having SEQ ID NO:2 The amino acid sequence is the position of the parent glucoamylase amino acid sequence. 3.权利要求1或2的变体，其中亲本葡糖淀粉酶具有与SEQ ID NO：2之氨基酸序列同一性至少约65％，优选至少约70％，更优选至少约80％，更优选至少约90％，最优选至少约95％，还最优选至少约97％的氨基酸序列。3. The variant of claim 1 or 2, wherein the parent glucoamylase has at least about 65% amino acid sequence identity with SEQ ID NO: 2, preferably at least about 70%, more preferably at least about 80%, more preferably at least About 90%, most preferably at least about 95%, and most preferably still at least about 97% amino acid sequence. 4.权利要求1-3的变体，其中的亲本葡糖淀粉酶为能在极低严谨条件下与SEQ ID NO：1的核酸序列或互补链杂交的核苷酸序列编码。4. The variant of claims 1-3, wherein the parent glucoamylase is encoded by a nucleotide sequence capable of hybridizing to the nucleotide sequence of SEQ ID NO: 1 or the complementary strand under extremely low stringency conditions. 5.权利要求1-4任一项的变体，其中亲本葡糖淀粉酶从曲霉属得到，特别是黑曲霉，或踝节菌属，特别是Talaromyces emersonii。5. The variant according to any one of claims 1-4, wherein the parent glucoamylase is obtained from Aspergillus, especially Aspergillus niger, or Talaromyces, especially Talaromyces emersonii. 6.权利要求1-5任一项的变体，其中亲本葡糖淀粉酶是从黑曲霉得到的黑曲霉G1或G2葡糖淀粉酶。6. The variant of any one of claims 1-5, wherein the parent glucoamylase is an Aspergillus niger G1 or G2 glucoamylase obtained from Aspergillus niger. 7.权利要求1-6任一项的变体，其中的改变是取代。7. The variant according to any one of claims 1-6, wherein the alteration is a substitution. 8.权利要求1-7任一项的变体，其中的改变是插入。8. The variant of any one of claims 1-7, wherein the alteration is an insertion. 9.权利要求1-8任一项的变体，其中的改变是缺失。9. The variant of any one of claims 1-8, wherein the alteration is a deletion. 10.权利要求1-9任一项的变体，其中的变体相对于亲本葡糖淀粉酶具有改进的热稳定性。10. The variant of any one of claims 1-9, wherein the variant has improved thermostability relative to the parent glucoamylase. 11.权利要求1-10任一项的变体，其中的变体相对于亲本葡糖淀粉酶具有提高的比活。11. The variant of any one of claims 1-10, wherein the variant has increased specific activity relative to the parent glucoamylase. 12.含有编码权利要求1-11任一项的葡糖淀粉酶变体的DNA序列的DNA构建体。12. A DNA construct comprising a DNA sequence encoding a glucoamylase variant according to any one of claims 1-11. 13.带有权利要求12的DNA构建体的重组表达载体。13. A recombinant expression vector carrying the DNA construct of claim 12. 14.用权利要求12的DNA构建体或权利要求13的载体转化的细胞。14. A cell transformed with the DNA construct of claim 12 or the vector of claim 13. 15.权利要求14的细胞，它是微生物，特别是细菌或真菌。15. The cell according to claim 14, which is a microorganism, in particular a bacterium or a fungus. 16.权利要求18的细胞，它是曲霉属菌株，特别是黑曲霉菌株。16. The cell according to claim 18, which is a strain of Aspergillus, especially a strain of Aspergillus niger. 17.权利要求17-19的细胞，它是踝节菌属菌株，特别是Talaromycesemersonii的菌株。17. The cell according to claims 17-19, which is a Talaromycesemersonii strain, in particular a Talaromycesemersonii strain. 18.将淀粉转化或部分水解为含葡萄糖的糖浆的方法，该方法包括在权利要求1-11任一项的葡糖淀粉酶变体存在下糖化淀粉水解产物的步骤。18. A process for the conversion or partial hydrolysis of starch to a glucose-containing syrup comprising the step of saccharifying a starch hydrolyzate in the presence of a glucoamylase variant according to any one of claims 1-11. 19.权利要求18的方法，其中葡糖淀粉酶变体的用量范围为0.05-0.5AGU/g干物质。19. The method of claim 18, wherein the glucoamylase variant is used in an amount in the range of 0.05-0.5 AGU/g dry matter. 20.权利要求18或19的方法，其中包含至少30％干重的淀粉水解产物的糖化。20. The method of claim 18 or 19, comprising saccharification of at least 30% dry weight of the starch hydrolyzate. 21.权利要求18-20任一项的方法，其中在选自支链淀粉酶和异淀粉酶组的脱支酶的存在下进行糖化，所述脱支酶优选得自Bacillusacidopullulyticus或Bacillus deramificans的支链淀粉酶或得自淀粉皮假单胞菌的异淀粉酶。21. The method of any one of claims 18-20, wherein saccharification is carried out in the presence of a debranching enzyme selected from the group of pullulanase and isoamylase, preferably derived from the branch of Bacillus acidopullulyticus or Bacillus deramificans Amylase or isoamylase from Pseudomonas amylodermis. 22.权利要求18-21任一项的方法，其中糖化条件为：pH3-5.5，温度60-80℃，优选63-75℃，进行24-72小时，优选在pH4-4.5进行36-48小时。22. The method according to any one of claims 18-21, wherein the saccharification conditions are: pH 3-5.5, temperature 60-80°C, preferably 63-75°C, for 24-72 hours, preferably at pH 4-4.5 for 36-48 hours . 23.权利要求1-11任一项的葡糖淀粉酶变体在淀粉转化过程中的应用。23. Use of the glucoamylase variant according to any one of claims 1-11 in a starch conversion process. 24.权利要求1-11任一项的葡糖淀粉酶变体在连续淀粉转化过程中的应用。24. Use of the glucoamylase variant according to any one of claims 1-11 in a continuous starch conversion process. 25.权利要求1-11任一项的葡糖淀粉酶变体在生产寡糖过程中的应用。25. Use of the glucoamylase variant according to any one of claims 1-11 in the production of oligosaccharides. 26.权利要求1-11任一项的葡糖淀粉酶变体在生产麦芽糖糊精或葡萄糖糖浆过程中的应用。26. Use of the glucoamylase variant according to any one of claims 1-11 in the production of maltodextrin or glucose syrup. 27.权利要求1-11任一项的葡糖淀粉酶变体在生产燃用或食用醇类过程中的应用。27. Use of the glucoamylase variant according to any one of claims 1-11 in the production of fuel or edible alcohols. 28.权利要求1-11任一项的葡糖淀粉酶变体在生产饮料中的应用。28. Use of the glucoamylase variant according to any one of claims 1-11 for the production of beverages. 29.权利要求1-11任一项的葡糖淀粉酶变体在生产有机化合物，例如柠檬酸，抗坏血酸，赖氨酸，谷氨酸的发酵过程中的应用。29. Use of the glucoamylase variant according to any one of claims 1-11 in a fermentation process for the production of organic compounds such as citric acid, ascorbic acid, lysine, glutamic acid. 30.通过将权利要求1-11中设定的一或多个位置改变从而改进亲本葡糖淀粉酶热稳定性和/或比活的方法。30. A method for improving the thermostability and/or specific activity of a parental glucoamylase by altering one or more of the positions set forth in claims 1-11.
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104870631A (en) * 2012-12-11 2015-08-26 丹尼斯科美国公司 Trichoderma reesei host cells expressing glucoamylases from Aspergillus fumigatus and methods of use thereof
CN109295032A (en) * 2010-07-01 2019-02-01 杜邦营养生物科学有限公司 Glucoamylase variant
CN110651048A (en) * 2017-04-11 2020-01-03 诺维信公司 Glucoamylase variants and polynucleotides encoding them
CN111032865A (en) * 2017-05-05 2020-04-17 希-莱科塔有限公司 Glucose isomerase

Families Citing this family (112)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6352851B1 (en) * 1998-07-15 2002-03-05 Novozymes A/S Glucoamylase variants
EP1200566A2 (en) * 1999-07-09 2002-05-02 Novozymes A/S Glucoamylase variant
AU2001278415A1 (en) 2000-08-01 2002-02-13 Novozymes A/S Alpha-amylase mutants with altered stability
ATE449840T1 (en) 2001-05-15 2009-12-15 Novozymes As ALPHA-AMYLASE VARIANT WITH MODIFIED PROPERTIES
EP1434861A2 (en) 2001-10-01 2004-07-07 Novozymes A/S Glucoamylase variants
JP4897186B2 (en) * 2002-03-27 2012-03-14 花王株式会社 Mutant alkaline cellulase
CN100412191C (en) 2002-12-17 2008-08-20 诺和酶股份有限公司 Thermostable alpha-amylase
WO2004106533A1 (en) 2003-05-30 2004-12-09 Novozymes A/S Alcohol product processes
ATE408674T1 (en) 2003-07-07 2008-10-15 Danisco As Danisco Intellectua VARIANTS OF NON-MALTOGENIC EXOAMYLASE FROM PSEUDOMONAS COMPREHENSIVE FOOD ADDITIVE
US8143048B2 (en) 2003-07-07 2012-03-27 Danisco A/S Exo-specific amylase polypeptides, nucleic acids encoding those polypeptides and uses thereof
ATE457034T1 (en) * 2003-10-28 2010-02-15 Novozymes North America Inc HYBRID ENZYMES
DE102004026152A1 (en) * 2004-05-28 2005-12-15 Basf Ag Fermentative production of fine chemicals
JP2008505632A (en) 2004-07-07 2008-02-28 ダニスコ　エイ／エス Non-maltogenic exoamylase variant
MX2007007107A (en) 2004-12-22 2007-08-08 Novozymes North America Inc Polypeptides having glucoamylase activity and polynucleotides encoding same.
US8030050B2 (en) 2005-07-07 2011-10-04 Danisco A/S Modified amylases from Pseudomonas species
US8216817B2 (en) 2005-09-20 2012-07-10 Novozymes North America, Inc. Process of producing a fermentation product
US20080318284A1 (en) 2005-12-22 2008-12-25 Novozymes North America, Inc. Processes for Producing a Fermentation Product
CN100443589C (en) * 2005-12-30 2008-12-17 湖南鸿鹰祥生物工程股份有限公司 Production of beta-amylase concentrated liquid with high conversion-rate and purity
US8076112B2 (en) 2006-03-22 2011-12-13 Novozymes A/S Fermentation processes
MX345513B (en) 2006-06-19 2017-02-01 Dupont Nutrition Biosci Aps Polypeptide.
JP5568307B2 (en) 2006-10-10 2014-08-06 ダニスコ・ユーエス・インク、ジェネンコー・ディビジョン Glucoamylase variants with modified properties
EP3219804B1 (en) 2007-04-24 2019-07-17 Novozymes North America, Inc. Detoxifying pre-treated lignocellulose-containing materials
WO2009030713A1 (en) 2007-09-03 2009-03-12 Novozymes A/S Detoxifying and recycling of washing solution used in pretreatment of lignocellulose-containing materials
US8592194B2 (en) 2007-10-09 2013-11-26 Danisco Us Inc. Glucoamylase variants with altered properties
EP2479265B1 (en) * 2007-10-09 2014-06-11 Danisco US Inc. Glucoamylase variants with altered properties
ES2527586T3 (en) * 2007-11-20 2015-01-27 Danisco Us Inc. Glucoamylase variants with modified properties
WO2009129086A2 (en) * 2008-04-18 2009-10-22 New England Biolabs, Inc. Endoglycosidases that cleave o-linked glycans
DK2447361T3 (en) 2008-06-06 2015-01-05 Danisco Us Inc Alpha-amylase (AMYS) variants of Geobacillus stearothermophilus with improved properties
CN102083991A (en) 2008-06-23 2011-06-01 诺维信公司 Processes for producing fermentation products
US20110165617A1 (en) 2008-09-30 2011-07-07 Novozymes North America, Inc. Enzymatic Hydrolysis Of Pretreated Lignocellulose-Containing Material With Distillers Dried Grains
EP2358878B1 (en) 2008-11-20 2014-10-15 Novozymes Inc. Polypeptides having amylolytic enhancing activity and polynucleotides encoding same
WO2010078391A2 (en) 2008-12-30 2010-07-08 Novozymes North America, Inc. Improvement of enzymatic hydrolysis of pretreated lignocellulose-containing material with dissolved air flotation sludge
WO2010078392A2 (en) 2008-12-31 2010-07-08 Novozymes North America, Inc. Processes of producing fermentation products
EP2451961A1 (en) 2009-07-07 2012-05-16 Novozymes A/S Process for treating a substrate with an enzyme
EP2454590B1 (en) 2009-07-17 2016-09-07 Novozymes A/S A method of analyzing cellulose decay in cellulosic material hydrolysis
CN102665425A (en) 2009-09-30 2012-09-12 诺维信公司 Steamed bread preparation methods and steamed bread improving compositions
EP2515931A4 (en) 2009-12-22 2013-05-22 Novozymes As COMPOSITIONS COMPRISING A REINFORCING POLYPEPTIDE AND A STARCH DEGRADING ENZYME, AND USES THEREOF
US20130040354A1 (en) 2010-01-29 2013-02-14 Novozymes A/S Biogas Production Process With Enzymatic Pre-Treatment
WO2011100161A1 (en) 2010-02-09 2011-08-18 Novozymes North America, Inc. Addition of alpha - glucosidase and cobalt for producing fermentation products from starch
ES2636216T3 (en) 2010-03-30 2017-10-05 Novozymes North America, Inc. Production processes of a fermentation product
ES2565060T3 (en) 2010-04-14 2016-03-31 Novozymes A/S Polypeptides having glucoamylase activity and polynucleotides encoding them
EP2579727B1 (en) 2010-06-11 2018-08-08 Novozymes A/S Enzymatic flour correction
US9308064B2 (en) 2010-07-26 2016-04-12 Johnson & Johnson Consumer Inc. Devices and methods for collecting and analyzing fluid samples from the oral cavity
CN103140586A (en) 2010-08-02 2013-06-05 诺维信北美公司 Process of producing a fermentation product
EP2640840B1 (en) 2010-11-19 2018-05-30 Novozymes North America, Inc. Process for producing a fermentation product employing an amino acid oxidase, an arginase and/or an asparaginase
CA2822637C (en) 2010-12-22 2020-06-30 Novozymes North America, Inc. Processes for producing fermentation products
CN103339260A (en) 2011-01-04 2013-10-02 诺维信公司 Process for producing biogas from pectin and lignocellulose containing materials
WO2012109119A2 (en) 2011-02-07 2012-08-16 Novozymes North America, Inc. Process of producing a fermentation product
MX2013014236A (en) 2011-06-28 2014-01-23 Novozymes As Biogas from enzyme-treated bagasse.
WO2013001078A1 (en) 2011-06-30 2013-01-03 Novozymes A/S Alpha-amylase variants
WO2013006756A2 (en) 2011-07-06 2013-01-10 Novozymes A/S Alpha amylase variants and polynucleotides encoding same
DK2734633T3 (en) 2011-07-22 2019-06-11 Novozymes North America Inc PROCEDURES FOR PREPARING CELLULOSE MATERIALS AND IMPROVING HYDROLYSIS THEREOF
US9128079B2 (en) 2011-08-08 2015-09-08 The Coca-Cola Company Methods of using lung or bronchial epithelial cells to identify bitter taste modulators
CN103987850B (en) 2011-10-11 2021-10-22 诺维信北美公司 Methods for producing fermentation products
EA201491087A1 (en) 2011-12-02 2014-09-30 Новозимс А/С METHODS OF PRODUCING ENERGY PRODUCTS
WO2013083801A2 (en) 2011-12-09 2013-06-13 Novozymes A/S Biogas from substrates comprising animal manure and enzymes
EP2794899A1 (en) 2011-12-21 2014-10-29 Novozymes, Inc. Methods for determining the degradation of a biomass material
CN104334738B (en) 2012-03-30 2019-01-29 诺维信北美公司 Methods of producing fermentation products
ES2935920T3 (en) 2012-03-30 2023-03-13 Novozymes North America Inc Production processes of fermentation products
CN104540394A (en) 2012-08-17 2015-04-22 诺维信公司 Thermostable asparaginase variants and polynucleotides encoding same
EP2893009A4 (en) 2012-09-07 2016-03-02 Novozymes As GLUCOMAMYLASE VARIANTS AND POLYNUCLEOTIDES ENCODING THE SAME AND USES THEREOF
DK3022300T3 (en) 2013-07-17 2018-10-22 Novozymes As : Pullulan chimeras and polynucleotides encoding them
WO2015035914A1 (en) 2013-09-11 2015-03-19 Novozymes A/S Processes for producing fermentation products
DK3415624T3 (en) 2014-01-22 2021-11-08 Novozymes As Pullulanase variants and polynucleotides that encode them
US10337041B2 (en) 2014-02-07 2019-07-02 Novozymes A/S Compositions for producing glucose syrups
WO2015143144A1 (en) 2014-03-19 2015-09-24 Novozymes A/S Method for enhancing activity of an x143 polypeptide
EP3550015B1 (en) 2014-04-10 2021-11-10 Novozymes A/S Alpha-amylase variants and polynucleotides encoding same
JP6548679B2 (en) 2014-07-08 2019-07-24 キャラヴァン イングリーディエンツ インク． Bakery method for producing sugar and improving texture and product formed therefrom
ES2743515T3 (en) 2014-10-23 2020-02-19 Novozymes As Variants of glucoamylase and polynucleotides encoding them
BR112017011275A2 (en) 2014-12-01 2018-04-03 Novozymes A/S composition, and process for the production of glucose syrup.
WO2016205127A1 (en) 2015-06-18 2016-12-22 Novozymes A/S Polypeptides having trehalase activity and the use thereof in process of producing fermentation products
EP3394273A1 (en) 2015-12-22 2018-10-31 Novozymes A/S Process of extracting oil from thin stillage
US10067091B2 (en) 2016-07-29 2018-09-04 Saudi Arabian Oil Company Integrated sediment and water analysis device and method
WO2018075430A1 (en) 2016-10-17 2018-04-26 Novozymes A/S Methods of reducing foam during ethanol fermentation
US10889836B2 (en) 2016-11-23 2021-01-12 Novozymes A/S Yeast for ethanol production
EP3630989A1 (en) 2017-06-02 2020-04-08 Novozymes A/S Improved yeast for ethanol production
EP4015524A1 (en) 2017-06-28 2022-06-22 Novozymes A/S Polypeptides having trehalase activity and polynucleotides encoding same
CN111094562A (en) 2017-08-08 2020-05-01 诺维信公司 Polypeptides with trehalase activity and their use in methods for producing fermentation products
CN111164214A (en) 2017-09-15 2020-05-15 诺维信公司 Enzyme blends and methods for improving the nutritional quality of animal feed
MX2020003981A (en) 2017-10-23 2020-08-03 Novozymes As Processes for reducing lactic acid in a biofuel fermentation system.
WO2019148192A1 (en) 2018-01-29 2019-08-01 Novozymes A/S Microorganisms with improved nitrogen utilization for ethanol production
AU2019222480B9 (en) 2018-02-15 2024-11-28 Microbiogen Pty. Ltd. Improved yeast for ethanol production
MX2020012754A (en) 2018-05-31 2021-02-26 Novozymes As Processes for enhancing yeast growth and productivity.
MX2021000112A (en) 2018-07-11 2021-03-09 Novozymes As Processes for producing fermentation products.
US11866751B2 (en) 2018-07-25 2024-01-09 Novozymes A/S Yeast expressing a heterologous alpha-amylase for ethanol production
CA3114783A1 (en) 2018-10-08 2020-04-16 Novozymes A/S Enzyme-expressing yeast for ethanol production
US11622751B2 (en) 2018-12-19 2023-04-11 Johnson & Johnson Consumer Inc. Devices and methods for collecting saliva samples from the oral cavity
CN113302294A (en) 2019-01-31 2021-08-24 诺维信公司 Polypeptides having xylanase activity and their use for improving the nutritional quality of animal feed
WO2020206058A1 (en) 2019-04-02 2020-10-08 Novozymes A/S Process for producing a fermentation product
CA3146541A1 (en) 2019-07-09 2021-01-14 Dupont Nutrition Biosciences Aps Fat coated particulate enzyme compositions
CN114450390A (en) 2019-07-26 2022-05-06 诺维信公司 Microorganisms with enhanced nitrogen transport for ethanol production
US20220279818A1 (en) 2019-08-05 2022-09-08 Novozymes A/S Enzyme blends and processes for producing a high protein feed ingredient from a whole stillage byproduct
BR112021026477A2 (en) 2019-08-06 2022-02-08 Novozymes As Recombinant host cell, methods of producing a fermentation product from a starch-containing or cellulose-containing material, of producing the mature polypeptide, of producing a derivative of a recombinant host cell, and of producing ethanol, nucleic acid construct or expression vector, composition and use of a recombinant host cell
WO2021046073A1 (en) 2019-09-05 2021-03-11 Dupont Nutrition Biosciences Aps Feed composition
MX2022002834A (en) 2019-09-16 2022-04-06 Novozymes As POLYPEPTIDES WITH BETA-GLUCANASE ACTIVITY AND POLYNUCLEOTIDES THAT ENCODE THEM.
CA3158982A1 (en) 2019-12-10 2021-06-17 Monica TASSONE Microorganism for improved pentose fermentation
US20230023446A1 (en) 2019-12-16 2023-01-26 Novozymes A/S Processes for producing fermentation products
WO2021163030A2 (en) 2020-02-10 2021-08-19 Novozymes A/S Polypeptides having alpha-amylase activity and polynucleotides encoding same
WO2022090564A1 (en) 2020-11-02 2022-05-05 Novozymes A/S Glucoamylase variants and polynucleotides encoding same
MX2023014545A (en) 2021-06-07 2024-03-01 Novozymes As Engineered microorganism for improved ethanol fermentation.
EP4408186A1 (en) 2021-09-27 2024-08-07 International N&H Denmark ApS Feed additive compositions and methods for using the same
US20250236858A1 (en) * 2021-10-15 2025-07-24 Danisco Us Inc. Glucoamylase variants and methods for use thereof
US20250319165A1 (en) 2022-05-17 2025-10-16 International N&H Denmark Aps Feed additive comprising enzyme combinations
EP4638725A1 (en) 2022-12-19 2025-10-29 Novozymes A/S Carbohydrate esterase family 3 (ce3) polypeptides having acetyl xylan esterase activity and polynucleotides encoding same
WO2024137252A1 (en) 2022-12-19 2024-06-27 Novozymes A/S Process for reducing syrup viscosity in the backend of a process for producing a fermentation product
EP4638768A2 (en) 2022-12-19 2025-10-29 Novozymes A/S Processes for producing fermentation products using fiber-degrading enzymes with engineered yeast
CN120380140A (en) 2022-12-19 2025-07-25 诺维信公司 Carbohydrate esterase family 1 (CE 1) polypeptides having feruloyl esterase and/or acetylxylan esterase activity and polynucleotides encoding same
CN120693401A (en) 2022-12-19 2025-09-23 诺维信公司 Compositions comprising arabinofuranosidase and xylanase and their use for increasing the solubility of hemicellulose fibers
WO2024227153A1 (en) 2023-04-28 2024-10-31 International N&H Denmark Aps Ruminant feed additive compositions
AU2024303961A1 (en) 2023-06-13 2025-12-11 Novozymes A/S Processes for producing fermentation products using engineered yeast expressing a beta-xylosidase
WO2025128568A1 (en) 2023-12-11 2025-06-19 Novozymes A/S Composition and use thereof for increasing hemicellulosic fiber solubilization
WO2026008449A2 (en) 2024-07-04 2026-01-08 Novozymes A/S A process for producing a fermentation product and a concentrated protein co-product

Family Cites Families (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4272326A (en) 1980-01-24 1981-06-09 Allied Chemical Corporation Enhanced distillation of cyclohexanol from phenol with additional cyclohexanone feed
US4760025A (en) 1984-05-29 1988-07-26 Genencor, Inc. Modified enzymes and methods for making same
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
DK122686D0 (en) 1986-03-17 1986-03-17 Novo Industri As PREPARATION OF PROTEINS
JPS62272987A (en) 1986-05-22 1987-11-27 Hitachi Plant Eng & Constr Co Ltd Production of glucose
DK463887D0 (en) 1987-09-07 1987-09-07 Novo Industri As GAERLEADER
JPH01191693A (en) 1988-01-25 1989-08-01 Tdk Corp Continuous saccharification of starch
CA1333777C (en) 1988-07-01 1995-01-03 Randy M. Berka Aspartic proteinase deficient filamentous fungi
JP2733330B2 (en) 1989-08-31 1998-03-30 日本食品化工株式会社 Method for producing high-purity starch sugar
CA2038485A1 (en) 1990-03-23 1991-09-24 Donald K. Hadden Nanofiltration process for making dextrose
JP3110452B2 (en) 1990-05-09 2000-11-20 ノボ ノルディスク アクティーゼルスカブ Cellulase preparation comprising endoglucanase enzyme
US5162210A (en) * 1990-06-29 1992-11-10 Iowa State University Research Foundation Process for enzymatic hydrolysis of starch to glucose
US5231017A (en) 1991-05-17 1993-07-27 Solvay Enzymes, Inc. Process for producing ethanol
US5665585A (en) * 1992-09-03 1997-09-09 Alko-Yhiot Oy Recombinant production of glucoamylase P in trichoderma
US5605793A (en) 1994-02-17 1997-02-25 Affymax Technologies N.V. Methods for in vitro recombination
DE4422198C2 (en) 1994-06-24 1997-08-28 Audi Ag Method for controlling the electrical heating of a catalytic converter
DK0894126T3 (en) 1996-03-27 2006-06-12 Novozymes As Alkaline phosphatase-deficient filamentous fungus
JP2000515377A (en) * 1996-07-24 2000-11-21 アイオワ ステイト ユニヴァーシティ リサーチ ファウンデーション インコーポレイテッド Protein manipulation of glucoamylase to increase pH optimum, substrate specificity and thermostability
ATE421578T1 (en) 1997-11-26 2009-02-15 Novozymes As THERMOSTABLE GLUKOAMYLASE
US6255084B1 (en) * 1997-11-26 2001-07-03 Novozymes A/S Thermostable glucoamylase
US6352851B1 (en) * 1998-07-15 2002-03-05 Novozymes A/S Glucoamylase variants
JP2002531121A (en) * 1998-12-07 2002-09-24 ノボザイムス アクティーゼルスカブ Glucoamylase having N-terminal extension
US6329186B1 (en) * 1998-12-07 2001-12-11 Novozymes A/S Glucoamylases with N-terminal extensions
EP1200566A2 (en) * 1999-07-09 2002-05-02 Novozymes A/S Glucoamylase variant
EP1280919A2 (en) * 2000-04-28 2003-02-05 Novozymes A/S Lipolytic enzyme variants
ATE457034T1 (en) * 2003-10-28 2010-02-15 Novozymes North America Inc HYBRID ENZYMES
US7464662B2 (en) * 2004-01-28 2008-12-16 Tokyo Electron Limited Compact, distributed inductive element for large scale inductively-coupled plasma sources

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109295032A (en) * 2010-07-01 2019-02-01 杜邦营养生物科学有限公司 Glucoamylase variant
CN104870631A (en) * 2012-12-11 2015-08-26 丹尼斯科美国公司 Trichoderma reesei host cells expressing glucoamylases from Aspergillus fumigatus and methods of use thereof
CN110651048A (en) * 2017-04-11 2020-01-03 诺维信公司 Glucoamylase variants and polynucleotides encoding them
CN111032865A (en) * 2017-05-05 2020-04-17 希-莱科塔有限公司 Glucose isomerase
CN111032865B (en) * 2017-05-05 2024-03-26 希-莱科塔有限公司 Glucose isomerase

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